U.S. patent application number 10/632175 was filed with the patent office on 2005-02-03 for methods and apparatus for introducing liquids into microfluidic chambers.
Invention is credited to Barth, Phillip W., Yefchak, George E..
Application Number | 20050022895 10/632175 |
Document ID | / |
Family ID | 33565202 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050022895 |
Kind Code |
A1 |
Barth, Phillip W. ; et
al. |
February 3, 2005 |
METHODS AND APPARATUS FOR INTRODUCING LIQUIDS INTO MICROFLUIDIC
CHAMBERS
Abstract
The present invention is directed to methods and apparatus for
removing a gaseous bubble confined in a microvolume of liquid in a
chamber. A source of liquid, a barrier region and an exit region
are provided in fluid communication with the chamber. The source of
liquid has an energy potential as regards movement of the gaseous
bubble that is higher than the energy potential of the barrier
region, the barrier region has a higher energy potential than the
chamber, and the chamber has a higher energy potential than the
exit region. The energy potential is reduced within the chamber,
the source of liquid, the barrier region, and the exit region by an
amount such that the energy within the gaseous bubble is sufficient
to displace the gaseous bubble from the chamber through the barrier
region and out the exit region and to fill the chamber with the
liquid from the source.
Inventors: |
Barth, Phillip W.; (Portola
Valley, CA) ; Yefchak, George E.; (Santa Clara,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
Legal Department, DL429
Intellectual Property Administration
P.O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
33565202 |
Appl. No.: |
10/632175 |
Filed: |
July 30, 2003 |
Current U.S.
Class: |
141/5 |
Current CPC
Class: |
B01L 2200/0684 20130101;
B01D 19/00 20130101; B01L 3/502723 20130101; B01L 2200/0642
20130101; B01L 3/0268 20130101; Y10T 137/206 20150401; G01N
2035/1018 20130101 |
Class at
Publication: |
141/005 |
International
Class: |
B67C 003/00 |
Claims
What is claimed is:
1. A method for removing a gaseous bubble confined in a microvolume
of liquid in a chamber, said method comprising: (a) providing in
fluid communication with said chamber a source of said liquid and a
barrier region and an exit region wherein said source of said
liquid has an energy potential as regards movement of said gaseous
bubble that is higher than the energy potential of said barrier
region, said barrier region has a higher energy potential than said
chamber, and said chamber has a higher energy potential than said
exit region, and (b) reducing the energy potential within said
chamber, said source, said barrier region, and said exit region by
an amount such that the energy within said gaseous bubble is
sufficient to displace said gaseous bubble from said chamber
through said barrier region and out said exit region and to fill
said chamber with said liquid from said source.
2. A method according to claim 1 wherein said energy potential is
reduced by reducing ambient pressure surrounding said chamber.
3. A method according to claim 1 wherein said chamber comprises an
aperture having an energy potential greater than the energy
potential of said barrier region and said exit region.
4. A method according to claim 1 wherein said aperture is selected
from the group consisting of micropores and nanopores.
5. A method according to claim 3 wherein said chamber is part of a
microfluidic system.
6. A method according to claim 5 wherein said microfluidic system
is selected from the group consisting of droplet dispensing devices
and microdevices having artificial nanopores.
7. A method according to claim 3 wherein said exit region is sealed
subsequent to filling of said chamber with said liquid.
8. A method of introducing a liquid into a chamber and avoiding
formation of or removing a gaseous bubble therein, said method
comprising: (a) introducing said liquid into said chamber from a
source of said liquid wherein said source, a barrier region and an
exit region are in fluid communication with said chamber and
wherein said source of said liquid has an energy potential as
regards movement of said gaseous bubble that is higher than the
energy potential of said barrier region, said barrier region has a
higher energy potential than said chamber, and said chamber has a
higher energy potential than said exit region, and (b) reducing the
energy potential within said chamber, said source, said barrier
region, and said exit region by an amount sufficient that the
energy within said gaseous bubble is sufficient to displace said
gaseous bubble from said chamber through said barrier region and
out said exit region and to fill said chamber with said liquid from
said source.
9. A method according to claim 8 wherein said energy potential is
reduced by reducing ambient pressure surrounding said chamber.
10. A method according to claim 8 wherein said chamber comprises an
aperture having an energy potential greater than the energy
potential of said barrier region and said exit region.
11. A method according to claim 8 wherein said aperture is selected
from the group consisting of micropores and nanopores.
12. A method according to claim 10 wherein said chamber is part of
a microfluidic system.
13. A method according to claim 12 wherein said microfluidic system
is selected from the group consisting of droplet dispensing devices
and microdevices having artificial nanopores.
14. A method according to claim 10 wherein said exit region is
sealed subsequent to filling of said chamber with said liquid.
15. An apparatus comprising: (a) a chamber, (b) a source of liquid
in fluid communication with said chamber, (c) a barrier region in
fluid communication with said chamber, (d) an exit region in fluid
communication with said chamber, and (e) an aperture in a wall of
said chamber.
16. An apparatus according to claim 15 wherein said aperture is
selected from the group consisting of micropores and nanopores.
17. An apparatus according to claim 15 wherein said chamber is part
of a microfluidic system.
18. An apparatus according to claim 17 wherein said microfluidic
system is selected from the group consisting of droplet dispensing
devices and microdevices having artificial nanopores.
19. An apparatus according to claim 17 further comprising a means
for sealing said exit region.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to microfluidic systems, and
more particularly, to methods and apparatus for introducing and
distributing fluid in channels of a microfluidic system. More
particularly, the invention relates to filling microfluidic systems
with liquids in a manner such that no gaseous bubbles are present
in the system after filling, because such bubbles, if present,
degrade the performance of the system. The microfluidic systems
include, for example, microdroplet dispensing devices, microdevices
with artificial nanopores, and the like.
[0002] In the field of diagnostics and therapeutics, it is often
useful to attach species to a surface. One important application is
in solid phase chemical synthesis wherein initial derivatization of
a substrate surface enables synthesis of polymers such as
oligonucleotides and peptides on the substrate itself. Substrate
bound oligomer arrays, particularly oligonucleotide arrays, may be
used in screening studies for determination of binding affinity.
Modification, of surfaces for use in chemical synthesis has been
described. See, for example, U.S. Pat. No. 5,624,711 (Sundberg),
U.S. Pat. No. 5,266,222 (Willis) and U.S. Pat. No. 5,137,765
(Farnsworth).
[0003] The arrays may be microarrays created on the surface of a
substrate by in situ synthesis of biopolymers such as
polynucleotides, polypeptides, polysaccharides, etc., and
combinations thereof, or by deposition of molecules such as
oligonucleotides, cDNA and so forth. In general, arrays are
synthesized on a surface of a substrate or substrate by one of any
number of synthetic techniques that are known in the art. In one
approach, for example, the substrate may be one on which a single
array of chemical compounds is synthesized. Alternatively, multiple
arrays of chemical compounds may be synthesized on the substrate,
which is then diced, i.e., cut, into individual assay devices,
which are substrates that each comprise a single array, or in some
instances multiple arrays, on a surface of the substrate.
[0004] The in situ synthesis methods include those described in
U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as
WO 98/41531 and the references cited therein for synthesizing
polynucleotides (specifically, DNA). Such in situ synthesis methods
can be basically regarded as repeating at each spot the sequence
of: (a) deprotecting any previously deposited monomer so that it
can now link with a subsequently deposited protected monomer; and
(b) depositing a droplet of another protected monomer for linking.
Different monomers may be deposited at different regions on the
substrate during any one iteration so that the different regions of
the completed array will have different desired biopolymer
sequences. One or more intermediate further steps may be required
in each iteration, such as oxidation, capping and washing steps.
The deposition methods basically involve depositing biopolymers at
predetermined locations on a substrate, which are suitably
activated such that the biopolymers can link thereto. Biopolymers
of different sequence may be deposited at different regions of the
substrate to yield the completed array. Washing or other additional
steps may also be used. Reagents used in typical in situ synthesis
are water sensitive, and thus the presence of moisture should be
eliminated or at least minimized.
[0005] Similar technologies can be used for in situ synthesis of
biopolymer arrays, such as DNA oligomer arrays, on a solid
substrate. In this case, each oligomer is formed nucleotide by
nucleotide directly in the desired location on the substrate
surface. This process demands repeatable drop size and accurate
placement on the substrate.
[0006] As indicated above, one of the steps in the synthesis
process usually involves depositing small volumes or microdroplets
of liquid containing reagents for the synthesis, for example,
monomeric subunits or whole polynucleotides, onto to surface of a
support or substrate. In one approach, pulse-jet techniques are
employed in depositing small volumes of liquid for synthesis of
chemical compounds on the surface of substrates. For example,
arrays may be fabricated by depositing droplets from a pulse-jet in
accordance with known techniques. The pulse-jet includes piezo or
thermal jets. Given the above requirements of biopolymer array
fabrication, deposition using pulse-jet techniques is particularly
favorable. In particular, pulse-jet deposition has advantages that
include producing very small spot sizes. This allows high-density
arrays to be fabricated. Furthermore, the spot size is uniform and
reproducible. Since it is a non-contact technique, pulse-jet
deposition does not result in scratching or damaging the surface of
the support on which the arrays are synthesized. Pulse-jet
techniques have very high deposition rate, which facilitates rapid
manufacture of arrays.
[0007] However, a pulse jet deposition system used for fabricating
a biopolymer array, should meet a number of requirements. The
system should provide for reliable dispensing of the reagents and
avoid deposition errors that can ruin the array fabrication. One
requirement is that the presence of gaseous bubbles in the system
must be minimized, eliminated, or prevented because gaseous bubbles
present a problem of hydraulic compliance, which degrades system
performance. Specifically, the pulse jet head must be capable of
being loaded, or primed, with very small volumes of expensive DNA
solution in a manner that minimizes, eliminates, or prevents
gaseous bubbles without wasting that DNA solution in the priming
process. Further, if gaseous bubbles occur in the pulse jet
deposition system after the priming process, it must be possible to
minimize or eliminate such bubbles without wasting that DNA
solution in the process of minimization or elimination.
[0008] Considerable work is now underway to develop microfluidic
systems, particularly for performing chemical, clinical, and
environmental analysis of chemical and biological specimens. The
term microfluidic system refers to a system or device having a
network of chambers connected by channels, in which the channels
have microscale features, that is, features too small to examine
with the unaided eye, e.g., having at least one cross-sectional
dimension in the range from about 0.1 .mu.m to about 1 mm. Such
microfluidic systems are often fabricated using photolithography,
wet chemical etching, and other techniques similar to those
employed in the semiconductor industry. The resulting devices can
be used to perform a variety of sophisticated chemical and
biological analytical techniques.
[0009] Microfluidic systems have a number of advantages over
conventional chemical or physical laboratory techniques. For
example, such microfluidic systems are particularly well adapted
for analyzing small sample sizes, typically making use of samples
on the order of nanoliters and even picoliters. The substrates may
be produced at relatively low cost, and the channels can be
arranged to perform numerous specific analytical operations,
including mixing, dispensing, valving, reactions, detections,
electrophoresis, and the like. The analytical capabilities of such
microfluidic systems are generally enhanced by increasing the
number and complexity of network channels, reaction chambers, and
the like.
[0010] Efficient filling of a microfluidic system with liquid can
be problematic because gas bubbles such as air bubbles can be
trapped in the liquid flow path during introduction of the liquid
into the microfluidic system. Such bubbles are difficult to remove
from such systems. A number of approaches have been postulated for
reducing or eliminating bubble formation during filling of
microfluidic systems. For example, in one approach a piezoelectric
system for dispensing DNA reagents is filled using degassed or
deaerated liquids. The process begins with introducing a buffer
solution, which is then replaced with an expensive reagent liquid
containing a dissolved compound such as, for example, a DNA
reagent. The object of this two-step procedure is to avoid
introducing air bubbles into the flow path. However, the
requirement of flushing the buffer solution with the expensive
reagent liquid results in some waste of the expensive reagent
liquid as well as waste of user time. See, for example, U.S. Patent
Application Publication No. US 2002/0122748.
[0011] Recently, work has been conducted on microfluidic systems
incorporating artificially fabricated nanopores. A nanopore is a
hole through a membrane wherein the hole has a diameter less that
approximately 100 nanometers (nm). Naturally occurring nanopore
molecules can be found in the membranes of living cells. For
example, the naturally-occurring alpha-hemolysin nanopore is a
protein complex with a minimum internal diameter of 1.5 nm, which
has been used in a simple microfluidic system to detect the passage
of single-stranded oligonucleotide molecules. Artificially
fabricated nanopores with diameters on the order of 2 to 100 nm
have been fabricated by drilling holes in membranes of silicon
nitride or silicon dioxide using a focused ion beam (FIB), followed
by narrowing of the drilled hole using sculpting with a low-energy
argon beam.
[0012] The process for establishing a liquid ionic conducting path
through an artificially fabricated nanopore often presents
difficulties. Such a pore may comprise a hole about 2 nm to about
70 nm in diameter in a membrane such as, e.g., silicon nitride or
silicon dioxide, typically about 60 nm thick and about 50 .mu.m in
length and width. When such a pore is placed in a microfluidic
system and the system is filled with an ionic buffer solution of
potassium chloride (KCl), it is almost invariably found that an air
bubble blocks electrical ionic conduction through the pore.
[0013] One approach to establishing conduction through an
artificial nanopore is to first introduce a buffer solution of KCl
in water to the structure holding the artificial nanopore, then
place the system in a vacuum chamber and reduce the air pressure
below atmospheric pressure. In this way, it is hoped that any
trapped air bubbles in the system expand greatly and then leave the
system when air pressure is increased again to atmospheric
pressure. Unfortunately, this approach sometimes fails because,
when the air pressure is increased, the trapped air bubble may
return to its original position, leaving the nanopore blocked.
[0014] It is therefore desirable to provide improved structures,
systems, and methods that overcome or substantially mitigate the
problems set forth above. In particular, there exists a need in
relation to the filling of microfluidic systems such as, for
example, inkjet heads and artificial nanopore structures, for
apparatus and methods that will reliably remove a gaseous bubble
from a chamber without wasting liquids or time or both.
[0015] U.S. Pat. No. 6,360,775 (Barth, et al.) discloses a
switching device for controlling fluid motion. The device includes
a capillary filled with a first fluid into which a wall-confined
bubble of a second fluid is introduced to achieve a first switching
event. Capillary geometry and wetting properties provide a
pressure-related asymmetric energy potential distribution for
controlling the flow of the bubble, and the device is called an
asymmetric bubble chamber, or ABC. The bubble is initially trapped
in an energy potential well, and upon increase of its volume moves
from the well into a region of low energy potential to achieve a
second switching event. The first switching event may be blocking
of a fluid channel or reflection of an optical beam in an optical
crosspoint switch, while the second switching event may be
unblocking of a fluid channel or restoration of transmission of an
optical beam. The increase in bubble volume between the first and
second switching events can act as the stroke of a fluidic piston
to pump a volume of the first fluid within the capillary. The
device can be employed to thermally degas a liquid. The use of
large-magnitude geometry-related energy potentials permits rapid
cyclical operation of the device in a manner resistant to
mechanical shock.
SUMMARY OF THE INVENTION
[0016] In the present invention a trapped gaseous bubble is removed
from a microfluidic system by means of reducing selected energy
potentials of the system, where such energy potentials regard the
energetics of movement of the bubble, to levels below the energy of
the trapped bubble so that the bubble has enough energy to exit the
system. The removal of the bubble achieves the purpose of complete
liquid filling of the microfluidic system.
[0017] For purposes of description, the gaseous bubble is a bubble
comprising a vapor of the liquid in which the bubble occurs, a gas
or gas mixture other than the vapor of the liquid, or a combination
of a vapor of the liquid and another gas or gas mixture. The bubble
is considered to be trapped in a chamber region or "chamber." The
chamber is in fluid communication with regions that function as a
source of replacement fluid or "source", as an energy barrier
region or "barrier," and as an exit region or "exit". The source,
chamber, barrier, and exit regions each have distinct energy
potential properties with respect to one another arising from
differences in geometry, differences in construction materials,
differences in surface layers, differences in applied voltages,
which produce electrowetting effects, or a combination of one or
more of the above.
[0018] In one embodiment of the present invention, the energy
potentials each vary directly with the magnitude of ambient gas
pressure surrounding the microfluidic system. In this embodiment,
ambient pressure is reduced to a level below atmospheric pressure
by means of placing the microfluidic system in a vacuum chamber and
pumping out some of the ambient gas from the vacuum chamber. The
number of moles of gas within the gas bubble may be substantially
constant as is explained more fully below. As ambient pressure is
reduced, the bubble expands, eventually displacing the bubble from
the chamber, past the barrier, to the exit. Simultaneously with the
movement of the bubble, liquid flows from the source into the
chamber to leave the entire microfluidic system filled with
degassed (deaerated) liquid. Then, the vacuum chamber is refilled
with gas to return the ambient pressure to atmospheric pressure.
The bubble may be exhausted from the exit to the vacuum chamber or
vacuum manifold. Subsequently, one or more of the barrier and exit
regions may be plugged to prevent liquid from leaving the device in
an undesired manner. Accordingly, in the present invention the
energy potentials of the source, the chamber, the barrier and the
exit are decreased with respect to the energy of the bubble, the
energy of the bubble being nearly constant, so that the bubble is
removed.
[0019] The present invention differs from the method of Barth, et
al., supra. In the present methods a microfluidic chamber is filled
with a liquid by equalizing bubble pressure in the chamber with an
applied pressure. The disclosure of Barth, et al., did not
contemplate this method. In Barth, et al., the energy within a
bubble is increased with respect to the energy potential of a
source, a gate, a barrier and a drain to a level greater than the
energy potential of the barrier region so that the bubble moves to
achieve the desired switching. In the present invention the energy
potentials of a liquid source, a chamber microvolume, a barrier
region and an exit region of a chamber are decreased with respect
to the energy of the bubble where the energy of the bubble is
nearly constant, thus resulting in movement of the bubble out of
the chamber.
[0020] One embodiment of the present invention is a method for
removing a gaseous bubble confined in a microvolume of liquid in a
chamber. A source of liquid, a barrier region and an exit region
are provided in fluid communication with the chamber. The source of
liquid has an energy potential as regards movement of the gaseous
bubble that is higher than the energy potential of the barrier
region, the barrier region has a higher energy potential than the
chamber, and the chamber has a higher energy potential than the
exit region. The energy potentials of the chamber, the source of
liquid, the barrier region, and the exit region are reduced by an
amount such that the energy within the gaseous bubble is sufficient
to displace the gaseous bubble from the chamber through the barrier
region and out the exit region and to fill the chamber with the
liquid from the source.
[0021] Another embodiment of the present invention is a method of
introducing a liquid into a chamber by means of a procedure that
avoids the presence of a gaseous bubble at the end of the
procedure, whether or not any gaseous bubble occurs in the chamber
during the procedure. In this embodiment any possible gaseous
bubble can be considered a "virtual bubble," that is, a bubble
which may or may not actually occur but, if it occurs, is removed.
The liquid is introduced into the chamber from, a source of liquid.
The source of liquid, a barrier region and an exit region are in
fluid communication with the chamber. The source of liquid has an
energy potential as regards movement of the gaseous bubble that is
higher than the energy potential of the barrier region, the barrier
region has a higher energy potential than the chamber, and the
chamber has a higher energy potential than the exit region. The
energy potential is reduced within the chamber, the source of
liquid, the barrier region, and the exit region by an amount such
that the energy within the gaseous bubble is sufficient to displace
the gaseous bubble from the chamber through the barrier region and
out the exit region and to fill the chamber with the liquid from
the source.
[0022] Another embodiment of the present invention is an apparatus
comprising a chamber, a source of liquid in fluid communication
with the chamber, a barrier region in fluid communication with the
chamber, an exit region in fluid communication with the barrier
region, and an aperture in a wall of the chamber.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following figures are included to better illustrate the
embodiments of the apparatus and technique of the present
invention. The figures are not to scale and some features may be
exaggerated for the purpose of illustrating certain aspects or
embodiments of the present invention.
[0024] FIG. 1A is a wireframe view of one embodiment of the present
invention.
[0025] FIG. 1B is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at one point in time.
[0026] FIG. 1C is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0027] FIG. 1D is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0028] FIG. 1E is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0029] FIG. 1F is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0030] FIG. 1G is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0031] FIG. 1H is a cross-sectional view of the embodiment of FIG.
1A taken along section line 101 at another point in time.
[0032] FIG. 2A is a wireframe view of one embodiment of the present
invention.
[0033] FIG. 2B is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at one point in time.
[0034] FIG. 2C is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at another point in time.
[0035] FIG. 2D is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at another point in time.
[0036] FIG. 2E is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at another point in time.
[0037] FIG. 2F is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at another point in time.
[0038] FIG. 2G is a cross-sectional view of the embodiment of FIG.
2A taken along section line 201 at another point in time.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0039] As mentioned above, the present invention is directed to
filling of a chamber with liquid while removing or avoiding gaseous
bubbles that might otherwise result from such filling. The chamber
is usually part of a microfluidic system. The term "microfluidic
system" as used herein refers to a system or device having fluidic
conduit features that are difficult or impossible to see with the
naked eye, that is, having features on a scale of millimeters to
tenths of micrometers. The size of the chambers is dependent on the
particular application in which the chamber is used. Such chambers
are found in microdevices such as droplet dispensing devices,
devices with artificial nanopores, micro total analysis systems,
and so forth. The present invention has application to any chamber
that is to be filled with a liquid where the operation of the
device after filling may be deleteriously affected by the presence
of a gaseous bubble. The chambers may have internal volumes of
about 1 picoliter to about 50 microliters and may in certain
circumstances be larger or smaller than the aforementioned volumes.
The terms "filling" and "fill" are used herein to mean introducing
liquid into the chamber to occupy at least about 98% of the volume,
at least about 99% of the volume, usually about 100% of the volume,
of the chamber.
[0040] The materials from which the chambers and related components
may be fabricated are dependent on the particular environment or
use of the chamber, the nature of the liquid within the chamber,
the desired differences in energy potentials in accordance with the
present invention, the advantages and limitations of particular
fabrication techniques, and so forth. Materials include polymers,
plastics, resins, polysaccharides, silica or silica-based
materials, carbon, metals including metal alloys, metal oxides,
inorganic glasses, and so forth. Particular plastics finding use
include, for example, polyethylene, polypropylene, such as high
density polypropylene, polytetrafluoroethylene (PTFE), e.g.,
TEFLON.RTM., polymethylmethacrylate, polycarbonate, polyethylene
terephthalate, polystyrene or styrene copolymers, polyurethanes,
polyesters, polycarbonates, polyureas, polyamides,
polyethyleneamines, polyarylene sulfides, polysiloxanes,
polydimethylsiloxanes, polyimides, polyacetates, poly
etheretherketone (PEEK), and the like. Metals include, for example,
stainless steel, hastalloy, platinum, gold, silver, titanium, and
so forth.
[0041] The interior of the chamber, the barrier region, the exit
region, the source of liquid, and the like may be coated with a
material that functions to change the energy properties of the
surfaces of any of the above. The coating may be any of the
aforementioned materials placed on the surface of the material from
which the chamber or one or more of its components are
fabricated.
[0042] The present devices may be fabricated as unitary devices or
they may be constructed from several parts assembled into the
device. Apertures may be made in the chamber housing by laser
cutting, etching, piercing, drilling, punching, direct molding or
casting from a master with pins, and so forth.
[0043] Droplet dispensing devices usually comprise one or more
chambers, which are filled with liquid to be dispensed. Typically,
a chamber has at least one aperture or orifice, usually, one
aperture or orifice, which is a micropore and through which
droplets are dispensed. A micropore is a pore (or aperture or
orifice) that is small usually on the order of micrometers (or
micron scale) or less. The size of the micropore as it relates to
the present invention is usually about 2 .mu.m to about 50 .mu.m,
more usually, about 4 .mu.m to about 40 .mu.m. The chamber is in
fluid communication with a source of liquid, which may be contained
in one or more reservoirs that are connected to the chamber by
suitable conduits and valves. The droplet dispensing devices also
include a means for causing the droplet to be dispensed, for
example a piezoelectric driver element or a thermal driver
element.
[0044] A number of approaches have been developed for accurately
dispensing small drops of liquid and depositing them onto solid
substrates. For example, inkjet printers utilize piezoelectric
dispensers to dispense liquid drops at rates of up to at least
2,000 drops per second. In one such system (known as a continuous
device) a fluid under pressure issues from an orifice in a
dispenser while a piezoelectric crystal attached to the dispenser
induces pressure oscillations in the fluid causing the fluid stream
to break into drops after issuing from the dispenser. The drops
form in the presence of an electrostatic field and thus acquire an
electric charge. As the drops continue toward the substrate, they
pass through another electrostatic field, which interacts with
their acquired charge to deflect them to a desired location.
[0045] In another inkjet system fluid from a reservoir is fed into
a dispenser and a piezoelectric crystal directly or indirectly
coupled to the fluid responds to a voltage pulse to induce a volume
change in the dispenser, thus causing a drop of fluid to issue from
an orifice toward a substrate. In this type of dispenser (known as
a drop-on-demand device) a drop is formed only in response to a
predetermined voltage pulse.
[0046] In addition to using piezoelectric effects, inkjets may also
use heat to form and propel drops of fluid. Thermal inkjets heat a
fluid so rapidly that the fluid vaporizes. Rapid volumetric changes
provide the impetus for propelling drops of fluid or ink from the
dispenser. Bubble jet printers also function on similar
principals.
[0047] The aforementioned jetting systems have been adapted to
dispense liquid reagents to a surface for conducting chemical
reactions such as in the analysis of analytes, synthesis of
chemical compounds, and the like. For example, in the manufacture
of nucleic acid arrays, inkjets can be used to deposit nucleic
acids on the substrate surface. See, for example, U.S. Pat. No.
5,658,802. U.S. Pat. No. 5,338,688 describes the use of a
bubble-jet for similar applications. The present invention has
application in all of the above systems.
[0048] As mentioned above, microfluidic systems include
microdevices with nanopores, usually, artificial nanopores. The
term microfluidic system refers to a system or device having a
network of chambers connected by channels, in which the channels
have mesoscale dimensions, e.g., having at least one
cross-sectional dimension in the range from about 0.1 .mu.m to
about 500 .mu.m. Typically, a chamber has at least one aperture or
orifice, which is a nanopore, i.e., a small pore (or aperture or
orifice) on the order of nanometers (i.e., nanometer scale).
Materials in a liquid contained within the chamber are moved
through the nanopore. The size of the nanopore is usually about 0.5
nm to about 100 nm, more usually, about 1.5 nm to about 30 nm. In
one example, a microfluidic fluid delivery system may include a
microfluidic device having a fluid input. A fluid reservoir is
fluid communication with the fluid input. The aforementioned
devices may also include means for introducing liquids into the
devices as well as means for moving materials in the liquids within
the devices. The resulting devices can be used to perform a variety
of sophisticated chemical and biological analytical techniques.
[0049] In one approach, microfluidic systems are employed to
separate materials in a microchannel and move the materials through
microchannels. Moving the materials through microchannels is
possible by use of a fluid pressure difference and by use of
various electro-kinetic processes including electrophoresis,
electroosmotic flow, and electrokinetic pumping. Microfluidic
devices have been designed that are useful in performing high
throughput assays useful for biological and chemical screening
experiments. Glass, polymer, semiconductor, ceramic, and metallic
microfluidic devices comprising microfluidic channels and
microfluidic wells are now available. Continuous flow microfluidic
systems are useful, for example, in screening large numbers of
different compounds for their effects on a variety of chemical and
biochemical systems. The devices include a series of channels
fabricated on or within the devices. The devices also can include
reservoirs, fluidly connected to the channels, which can be used to
introduce a number of test compounds into the sample channels and
thus perform the assays. Interfacing mechanisms, such as
electropipettors, can be incorporated into these high-throughput
systems for transporting samples into wells or microfluidic
channels.
[0050] Microfluidic systems for fast, accurate and low cost
electrophoretic analysis of materials in the fields of chemistry,
biochemistry, biotechnology, molecular biology and numerous other
fields are described in U.S. Pat. No. 5,699,157. Techniques for
transporting materials through microfluidic channels using
electrokinetic forces are described in U.S. Pat. No. 5,799,868.
Movement of material through microfluidic channels is further
described in U.S. Pat. No. 5,800,690.
[0051] Regardless of the particular environment in which the
chamber is found, the benefits of the present invention are
realized by providing, in fluid communication with the chamber, a
source of fluid, a barrier region and an exit region. The barrier
region has energy potential as regards movement of the gaseous
bubble that is higher than the energy potential of the exit region
and higher than the energy potential of the chamber. The source of
liquid has a higher energy potential than the barrier region. The
chamber has a higher energy potential than the exit region.
Typically, a gaseous bubble is present in the chamber that is
preventing the chamber from filling with a predetermined volume of
liquid, usually, a microvolume of liquid that corresponds to the
capacity volume of the chamber. The gaseous bubble may present an
undesirable mechanical compliance to the ejection of liquid from
the chamber by pulsejet means. Alternatively, the gaseous bubble
may prevent an ionic electrical conduction path from being
established through a nanopore in fluid communication with the
chamber. On the other hand, the bubble may be interfering with the
passage of a material such as, for example, particles such as
charged particles, e.g., positive and negative ions, solid
particles present as a slurry in the liquid, molecules dissolved in
the liquid and the like, through an aperture that provides an exit
from the chamber other than the aforementioned exit region, for
example, through the firing orifice of an inkjet device. The bubble
is usually confined in the microvolume of liquid. It is important
to note that not all gaseous bubbles within one or more chambers of
a microfluidic device conflict with the ability to move materials
through an aperture. In the latter circumstance, it is not
necessary to remove such a bubble from a chamber.
[0052] The source, chamber, barrier, and exit regions each have
distinct energy potential properties with respect to one another
arising from differences in geometry, differences in construction
materials, differences surface layers, differences in applied
voltages that produce electrowetting effects, or a combination of
one or more of the above.
[0053] The barrier region is normally situated between the exit
region and the microvolume of fluid in which the gaseous bubble
resides such that the gaseous bubble enters the barrier region
before the exit region in a spatial sense. To achieve a difference
in energy potential as a result of a difference in material of
composition of the barrier region and the exit region, the
hydrophobicity or hydrophilicity of the materials or coatings on
the interior surfaces of the materials may be considered.
[0054] The source of liquid may be positioned in any area of the
chamber such that liquid is introduced into the chamber coincident
with the removal of a gaseous bubble therefrom. In one embodiment
the source of liquid is adjacent the barrier region and the exit
region. In another embodiment, the source of liquid is through the
barrier region and the exit region.
[0055] As mentioned above, the source of liquid has an energy
potential as regards movement of the gaseous bubble that is higher
than the energy potential of the barrier region, the barrier region
has a higher energy potential than the chamber, and the chamber has
a higher energy potential than the exit region.
[0056] A chamber may have one of many cross-sectional shapes such
as a square, rectangular, trapezoidal, circular, oval, etc., cross
section. Furthermore, the cross-section of the interior of a
chamber may have several different cross-sectional shapes. For
example, the cross-sectional shape of an area of the chamber
adjacent a pore or opening or orifice may be different than that of
the remainder of the chamber. A "candidate bubble" in a chamber is
a bubble that must be removed because the bubble is blocking
transport of materials through an opening in the chamber or is
preventing the filling of the chamber with liquid for expulsion
through an opening. Usually, at least a portion of the periphery of
the bubble is in contact with the walls of the chamber. Where the
bubble is preventing the transport of materials through an opening,
a portion of the periphery of the bubble is in contact with the
interior walls of the chamber immediately adjacent the opening.
Where the bubble is preventing filling of the chamber, the bubble
may be at any location within the chamber.
[0057] Between a fluid bubble such as a gas bubble and its fluid
surroundings such as a liquid, there exists an interfacial surface
which can be characterized by a radius of curvature r and a surface
tension .sigma. (T), where T is temperature and so .sigma. is a
function of temperature T. Across this surface there exists a
pressure difference given by P=2 .sigma.(T)/r (see, for example,
Physical Chemistry, Walter J. Moore, fourth edition, Prentice-Hall,
Englewood Cliffs, N.J., page 478).
[0058] The bubble surface can be manipulated by varying one or more
of the pressure difference, the surface tension, the surface radius
of curvature, and the wetting properties of the capillary
walls.
[0059] Good wetting and poor wetting can be quantified in terms of
equilibrium contact angles of fluid against a surface. For example,
a drop of water in air contacting a clean plate of silicon dioxide
glass has a very low equilibrium contact angle taken within the
water, and the glass surface is said to be well wetted. However,
the contact angle taken within the air is large, and so the air is
considered to "wet" poorly in comparison to water. On the other
hand, a drop of mercury in air resting on a clean glass plate has a
very high equilibrium contact angle taken within the mercury
droplet. The glass surface is said to be poorly wetted by the
mercury, and the air is considered to wet well in comparison to the
mercury. For aqueous liquids good wetting is called hydrophilicity
and is characterized by an equilibrium contact angle less than
ninety degrees; the wetted material is described as hydrophilic.
Similarly, poor wetting is called hydrophobicity and is
characterized by an equilibrium contact angle greater than ninety
degrees; the wetted material is described as hydrophobic. The terms
hydrophilic and hydrophobic can be generalized to "fluiphilic" and
"fluiphobic" to describe the equilibrium contact angle taken within
any fluid where it meets a second immiscible fluid at a solid
wall.
[0060] The energy potential of a region for a gaseous bubble in a
liquid in a chamber can be influenced both by geometry and by
temperature. For example, for a bubble of gas within a liquid that
is fluiphilic to the capillary walls of a liquid source, narrow
capillaries have a higher energy potential than wider capillaries,
and cooler regions have a higher energy potential than warmer
regions. The above are some of the factors that may be controlled
to achieve the differences in energy potential between the source
of liquid and the barrier region, the barrier region and the
chamber and the chamber and the exit region.
[0061] In the next step in accordance with the method of the
present invention, the energy potential is reduced within the
chamber, the source of liquid, the barrier region, and the exit
region by an amount sufficient that the energy within the gaseous
bubble is sufficient to displace the gaseous bubble from the
chamber through the barrier region and out the exit region and to
fill the chamber with the liquid from the source.
[0062] The energy contained within a bubble due to pressure is just
the internal pressure of the bubble with respect to its
surroundings multiplied by the volume of the bubble. Thus, the
pressure is one measure of the energy. Other factors such as
gravity and temperature can contribute their own energy.
[0063] Accordingly, one approach to reducing the energy potential
within the chamber, the source of liquid, the barrier region and
the exit region is to reduce hydrostatic pressure in these regions.
To this end, ambient pressure may be reduced by placing the
microfluidic system in a vacuum housing and applying a vacuum in a
continuous manner so as to reduce ambient pressure to a level below
that of the internal pressure of the bubble. Once the gaseous
bubble has been removed from the chamber through the exit region
the pressure surrounding the microfluidic system is returned to its
original level, usually, ambient level.
[0064] As mentioned above, in one embodiment of the present
invention, the energy potentials each vary directly with the
magnitude of ambient gas pressure surrounding the microfluidic
system. In this embodiment, ambient pressure is reduced to a level
below atmospheric pressure by means of placing the microfluidic
system in a vacuum chamber and pumping out some of the ambient gas
from the vacuum chamber. The number of moles of gas within the gas
bubble may be substantially constant, which may be explained more
fully as follows. As is well known, when the ambient pressure
surrounding a liquid is reduced, any gas dissolved in that liquid
tends to leave the liquid in accordance with Henry's Law as the
partial pressure of the gas in the ambient is reduced below the
partial pressure of the gas in the liquid. This process of gas
leaving the liquid can result in the generation of gaseous bubbles,
or in the enlargement of existing gaseous bubbles, either of which
events increases the number of moles of gas in a bubble and
increases the size of a gaseous bubble at constant ambient
pressure. However, such an increase in the number of moles of gas
in a bubble may be regarded as inadvertent and unavoidable as
regards the action of the present invention wherein the size of a
gaseous bubble increases due to a reduction in pressure while the
number of moles of gas in the bubble is substantially constant. In
any event the present invention works regardless of whether or not
additional gas enters a bubble.
[0065] As is also well known, the boiling temperature of a liquid
commonly decreases as the ambient pressure of gas surrounding the
liquid decreases. Thus, a liquid at room temperature can begin to
boil as the ambient pressure of the gas surrounding the liquid
decreases. This process of boiling due to reduction in ambient
pressure can result in the generation of gaseous bubbles, or in the
enlargement of existing gaseous bubbles, either of which events
increases the number of moles of vapor in a gaseous bubble and
increases the size of a gaseous bubble at constant ambient
pressure. However, such an increase in the number of moles of vapor
in a bubble may be regarded as inadvertent and unavoidable as
regards the action of the present invention wherein the size of a
gaseous bubble increases due to a reduction in pressure while the
number of moles of vapor in the bubble is substantially constant.
It is well known that when a liquid boils due to a reduction in
ambient pressure, the temperature of the liquid falls, and that
such a liquid eventually stops boiling in the absence of a further
input of thermal energy from its surroundings. In any event the
present invention works regardless of whether or not additional
vapor enters a bubble.
[0066] As ambient pressure is reduced in accordance with the
present invention, the bubble expands, eventually displacing the
bubble from the chamber, past the barrier, to the exit.
Simultaneously with the movement of the bubble, liquid flows from
the source into the chamber to leave the entire microfluidic system
filled with degassed (deaerated) liquid. Then, the vacuum chamber
is refilled with gas to return the ambient pressure to atmospheric
pressure. The bubble may be exhausted from the exit to the vacuum
chamber or vacuum manifold. Subsequently, one or more of the
barrier and exit regions may be plugged to prevent liquid from
leaving the device in an undesired manner. Accordingly, in the
present invention the energy potentials of the source, the chamber,
the barrier and the exit are decreased with respect to the energy
of the bubble, the energy of the bubble being nearly constant, so
that the bubble is removed.
[0067] FIGS. 1A-1H illustrate, by way of example and not
limitation, an embodiment 100 of the invention as it is applied to
priming an artificial nanopore. As mentioned above, FIG. 1A is a
wireframe view of embodiment 100, and section line 101 denotes the
cross section of embodiment 100 shown at sequential time steps in
FIG. 1B-1H. FIG. 1B corresponds in time to FIG. 1A, while FIG.
1C-1H correspond to subsequent times.
[0068] Nanopore 102 extends through freestanding window 104, which
forms part of layer 106. Layer 106 is surrounded by sloping
sidewalls 108 situated in silicon chip 110. Silicon chip 110 is
supported between housing members 112 and 114, which contain
passages 116 and 118, respectively, to form two chambers 115 and
117. Tapered tubes 120 and 122 are secured into place in passages
116 and 118 to aid in liquid filling and degassing. Tube 124, which
corresponds to a source of liquid, is used as part of the invention
to aid in filling passage 116 with liquid volume 126. Passage 118
is filled with liquid volume 128. Bubble 130 is a gaseous bubble
within liquid volume 126, and bubble 132 is a gaseous bubble within
liquid volume 128. Gaseous bubble 130 blocks the free flow of
particles through nanopore 102 and is held in place by adhesion
forces to window 104 and sidewalls 108. For the nanopore to
function as desired, bubble 130 must be removed. The structure and
method of the present invention are utilized to remove bubble
130.
[0069] Gaseous bubble 132 is so situated that it does not block the
free flow of particles through nanopore 102, and it is not
necessary to remove bubble 132; thus it is not necessary to
implement the structure and method of the present invention in
conjunction with features 114, 118, 122, 128, and 132 on the
right-hand side of FIG. 1B.
[0070] FIG. 1C illustrates embodiment 100 at a time subsequent to
the time of FIG. 1B. Embodiment 100 has been placed in a vacuum
housing, not shown, and ambient atmosphere 133 has been reduced in
pressure to a value below atmospheric pressure by pumping on the
vacuum housing using a vacuum pump, not shown. As ambient
atmosphere 133 is reduced in pressure, bubbles 130 and 132 expand
in volume.
[0071] FIG. 1D shows embodiment 100 at a time subsequent to the
time of FIG. 1C. Ambient atmosphere 133 has been further reduced in
pressure, and bubbles 130 and 132 have further expanded in volume.
Bubble 130 fills a volume 134. Channel 136 comprises a source
channel for liquid, narrow region 138 comprises a barrier region,
and channel 140 and ambient gaseous volume 142 comprise an exit
region.
[0072] FIG. 1E shows embodiment 100 at a time subsequent to the
time of FIG. 1D. Ambient atmosphere 133 has been further reduced in
pressure, and bubbles 130 and 132 have further expanded in volume.
Bubble 130 has expanded past the narrow barrier region 138, and the
radii of curvature of bubble boundary 144 are increasing as this
boundary moves up in channel 140.
[0073] FIG. 1F shows embodiment 100 at a time subsequent to the
time of FIG. 1E. Ambient atmosphere 133 has been further reduced in
pressure, and bubble 132 has expanded further in volume. But bubble
130 has burst at boundary 144, and the gaseous volume of bubble 130
exiting past barrier region 138 through the exit region comprising
channel 140 and ambient gaseous volume 142 as the gate volume 134
is refilled with liquid supplied by source channel 136.
[0074] FIG. 1G shows embodiment 100 at a time subsequent to the
time of FIG. 1F. Ambient atmosphere 133 has been increased to the
original atmospheric pressure. Bubble 130 has completely exited
embodiment 100, chamber 115 has filled with liquid, and bubble 132
has shrunk to its original size and returned to a position near its
original position. Because bubble 130 has been removed, nanopore
102 is no longer blocked.
[0075] FIG. 1H shows embodiment 100 at a time subsequent to the
time of FIG. 1G. Embodiment 100 has been removed from the vacuum
chamber, and tubes 120, 122, and 124 have been removed. Bubble 132
does not block nanopore 102, and access to the nanopore is
obtainable through channels 116 and 118.
[0076] FIGS. 2A-2G illustrate, by way of example and not
limitation, an embodiment 200 of the invention as it applies to
priming a piezoelectric inkjet firing chamber. As mentioned above,
FIG. 2A is a wireframe view of embodiment 200, and section line 201
denotes the cross section of embodiment 200 shown at sequential
time steps in FIGS. 2B-2G. FIG. 2B corresponds in time to FIG. 2A,
while FIGS. 2C-2G correspond to subsequent times.
[0077] In embodiment 200 as illustrated in FIGS. 2A and 2B, firing
chamber 202 comprises firing chamber volume 202a, which is
surrounded by firing chamber walls 203. Piezoelectric actuator 204
is adjacent to wall area 205 and, during normal operation of the
inkjet, causes ink to be ejected from firing orifice 206 to a
substrate, not shown, such as paper or glass. Liquid 207, for
example, ink, is supplied to firing chamber 202 through orifice 208
from source channel 210. For purposes of the present invention,
barrier orifice 212 and an exit region comprising aperture 214 and
ambient volume 215 are included with the inkjet firing chamber.
Region 216 may be occupied in part by gaseous bubble 218, which is
present in spite of careful filling procedures, or which originates
within the liquid 207 because of outgassing of dissolved gas.
Gaseous bubble 218 can prevent the inkjet from firing because it
introduces a gas elasticity to the system; the situation is similar
to the problem of air in a brake line of an automobile which can
cause the brakes to work poorly.
[0078] In conventional inkjet structures, which do not have
features 212, 214, and 215, removal of such a bubble is problematic
and would be attempted by flushing large quantities of ink from
source channel 210, through the firing chamber volume 202a, and out
the firing orifice 206, in the hope of carrying bubble 218 out of
the firing chamber volume 202a through the firing orifice 206. The
present invention avoids wasting ink and removes the bubble 218
through barrier region 212 and to the exit region comprising
aperture 214 and ambient volume 215. Firing orifice 206 corresponds
to an aperture for dispensing droplets of liquid from chamber 202.
It should be understood that the energy potential of such aperture
is higher than that of the barrier region and exit region so that
liquid does not exit the aperture during the bubble removal
process.
[0079] FIG. 2C illustrates embodiment 200 at a time subsequent to
the time of FIG. 2B. Embodiment 200 has been placed in a vacuum
chamber, not shown, and ambient pressure 219 has been reduced in
pressure to a value below atmospheric pressure by pumping on the
vacuum chamber using a vacuum pump, not shown. As ambient pressure
219 is reduced, bubble 218 expands in volume to fill the firing
chamber volume 202a.
[0080] FIG. 2D illustrates embodiment 200 at a time subsequent to
the time of FIG. 2C. Pressure 219 has been further reduced, and
bubble 218 has extruded through barrier orifice 212. Bubble
boundary 220 is attached at aperture 214, and extends outward into
ambient volume 215. As explained above, bubble 218 has not extruded
through orifice 206 or orifice 208 because those two orifices
present higher energy potential barriers to bubble extrusion than
does barrier orifice 212. Additional liquid has entered firing
chamber volume 202a through orifice 208 from source channel 210,
has crept past the edges of the bubble 218 where the bubble 218
fails to completely occupy the corners of the firing chamber volume
202a and has filled in the volume firing chamber volume 202a near
firing orifice 206 as the bubble moves from right to left to the
exit region comprising aperture 214 and ambient volume 215.
[0081] FIG. 2E illustrates embodiment 200 at a time subsequent to
the time of FIG. 2D. Pressure 219 has been further reduced, and
bubble 218 has burst. The remains of bubble boundary 220 are drawn
as free droplets 224 and fringe 226 located adjacent to aperture
214, but no attempt has been made to accurately portray the
bursting process. Bubble 218 is still in the process of exiting
from firing chamber 202 to ambient volume 215.
[0082] FIG. 2F illustrates embodiment 200 after the bubble has
exited firing chamber volume 202. The firing chamber volume 202a is
completely refilled with liquid 207 from source channel 210, and no
bubbles are present.
[0083] FIG. 2G illustrates embodiment 200 after a plug 228 has been
secured in aperture 214. The plug supplies sufficient rigidity to
the firing chamber so that mechanical pulses originating at
piezoelectric driver 204 can expel droplet of liquid 207 from
firing orifice 206.
[0084] Another embodiment of the present invention is an apparatus
comprising a chamber, a source of liquid in fluid communication
with the chamber, a barrier region in fluid communication with the
chamber, an exit region in fluid communication with the chamber,
and an aperture. The aperture may be selected from the group
consisting of micropores and nanopores. The chamber may be part of
a microfluidic system, which may be selected from the group
consisting of droplet dispensing devices and microdevices having
artificial nanopores. The apparatus may further include a plug for
sealing the exit region after removal of a bubble. The plug may be
attached by friction fitting, screw fitting, luer-lock fitting,
glue, solder, brazing, welding, compression fitting, clamp fitting,
and the like. The plug may be constructed any of a wide variety of
rigid or flexible materials including rubbers, elastomers, metals,
polymers, ceramics, glasses, and the like.
[0085] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference, except
insofar as they may conflict with those of the present application
(in which case the present application prevails). Methods recited
herein may be carried out in any order of the recited events, which
is logically possible, as well as the recited order of events.
[0086] The aforementioned description includes theories and
mechanisms by which the invention is thought to work. It should be
noted, however, that such proposed theories and mechanisms are not
required and the scope of the present invention should not be
limited by any particular theory and/or mechanism.
[0087] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Furthermore, the foregoing description, for purposes of
explanation, used specific nomenclature to provide a thorough
understanding of the invention. However, it will be apparent to one
skilled in the art that the specific details are not required in
order to practice the invention. Thus, the foregoing descriptions
of specific embodiments of the present invention are presented for
purposes of illustration and description; they are not intended to
be exhaustive or to limit the Invention to the precise forms
disclosed. Many modifications and variations are possible in view
of the above teachings. The embodiments were chosen and described
in order to explain the principles of the invention and its
practical applications and to thereby enable others skilled in the
art to utilize the invention.
* * * * *