U.S. patent application number 11/165749 was filed with the patent office on 2005-10-27 for control of operation conditions within fluidic systems.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Morrissey, Donald J. JR., Parce, J. Wallace, Yao, Yung-mae M..
Application Number | 20050238545 11/165749 |
Document ID | / |
Family ID | 26917411 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050238545 |
Kind Code |
A1 |
Parce, J. Wallace ; et
al. |
October 27, 2005 |
Control of operation conditions within fluidic systems
Abstract
Methods of controlling environmental conditions within a fluidic
system, where such environmental conditions can affect the
operation of the system in its desired function, and fluidic
channels, devices and systems that are used in practicing these
methods. Such methods are generally directed to environmental
control fluids, the movement of such fluids through these systems,
and the interaction of these fluids with other components of the
system, e.g., other fluids or solid components of the system.
Inventors: |
Parce, J. Wallace; (Palo
Alto, CA) ; Yao, Yung-mae M.; (Newtown, MA) ;
Morrissey, Donald J. JR.; (Redwood City, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
26917411 |
Appl. No.: |
11/165749 |
Filed: |
June 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11165749 |
Jun 23, 2005 |
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09993385 |
Nov 14, 2001 |
|
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09993385 |
Nov 14, 2001 |
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09919369 |
Jul 31, 2001 |
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60223072 |
Aug 4, 2000 |
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Current U.S.
Class: |
422/400 ;
204/451 |
Current CPC
Class: |
G01N 35/08 20130101;
B01L 2200/141 20130101; B01L 2400/0415 20130101; B01L 2400/049
20130101; B01L 3/502746 20130101; Y10T 137/0391 20150401; B01L
2400/084 20130101; B01L 2300/105 20130101; B01L 3/502784 20130101;
B01L 2200/0684 20130101; G01N 27/44791 20130101; G01N 2035/00237
20130101; B01L 3/5027 20130101; G01N 2035/1034 20130101; Y10T
137/0324 20150401; B01L 2300/14 20130101; B01L 2200/0673 20130101;
B01L 2400/0487 20130101; B01L 2300/0816 20130101; Y10T 436/2575
20150115 |
Class at
Publication: |
422/100 ;
204/451 |
International
Class: |
G01N 027/27 |
Claims
What is clamed is:
1. A method of using an environmental control reagent to maintain
optimal conditions within a microfluidic device, the method
comprising: introducing a volume of a first fluid into a channel
segment of a microfluidic device, the first fluid comprising an
environmental control reagent; and introducing a volume of a second
fluid into the channel segment of the microfluidic device, the
second fluid comprising a component of a reaction mixture.
2. The method of claim 1, wherein the volume of the first fluid is
introduced into the channel segment after the volume of the second
fluid is introduced into the channel segment.
3. The method of claim 1, further comprising introducing a volume
of a third fluid into the channel segment.
4. The method of claim 1, wherein introducing a volume of a first
fluid into a channel segment of a microfluidic device comprises
treating a surface of the channel segment.
5. The method of claim 4, wherein treating a surface of the channel
segment comprises one or more actions selected from a group
consisting of modifying the surface of the channel segment, coating
the surface of the channel segment, and cleaning the surface of the
channel segment.
6. The method of claim 4, wherein the first fluid includes one of a
monovalent or a bivalent compound.
7. The method of claim 4, wherein the first fluid includes a
surface adsorbing polymer.
8. The method of claim 7, wherein the surface adsorbing polymer is
selected from a group consisting of a linear cellulose polymer, an
agarose polymer, an acrylic polymer, a polyacrylamide, a linear
polyacrylamide polymer, a linear polyacrylamide copolymer, a linear
polydimethylacrylamide polymer, a linear polydimethylacrylamide
copolymer, and a combination thereof.
9. The method of claim 4, wherein the first fluid includes a
cleaning reagent.
10. The method of claim 9, wherein the cleaning reagent is selected
from a group consisting of an acid, a base, a detergent, a high
salt solution, a zwitterionic solution, and a combination
thereof.
11. The method of claim 1, wherein introducing a volume of a first
fluid into a channel segment of a microfluidic device comprises
altering flow resistance within the channel segment.
12. The method of claim 11, wherein the first fluid includes a
viscosity adjusting reagent.
13. The method of claim 12, wherein the viscosity adjusting reagent
is selected from a group consisting of a polymeric reagent, a
polysaccharide, a polysaccharide polymer, a polyacrylamide,
gelatin, and a combination thereof.
14. The method of claim 1, wherein introducing a volume of a first
fluid into a channel segment of a microfluidic device comprises
minimizing gas evolution within the channel segment.
15. The method of claim 14, wherein the first fluid is not gas
saturated.
16. The method of claim 14, wherein the first fluid is heated
immediately prior to introducing the first fluid into the channel
segment.
17. The method of claim 14, wherein the first fluid is subjected to
a negative pressure immediately prior to introducing the first
fluid into the channel segment.
18. The method of claim 1 wherein the first fluid includes a
signaling component.
19. The method of claim 18, wherein the signaling component is
selected from a group consisting of a dye, a label, a marker, and a
combination thereof.
20. The method of claim 18, wherein the signaling component is
selected from a group consisting of a temperature indicator, a pH
indicator, a conductivity indicator, and a combination thereof.
21. The method of claim 1, wherein the first fluid includes a
calibrator.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/993,385, filed Nov. 14, 2001, which is a
continuation-in-part of U.S. patent application Ser. No.
09/919,369, filed Jul. 31, 2001, which claims priority to
Provisional Patent Application No. 60/223,072, filed Aug. 4, 2000.
The full disclosure of each of these applications is incorporated
herein by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Microfluidic systems have advanced to the point where they
are beginning to supplant conventional technologies in biological,
chemical and biochemical analyses. For example, routine separation
based analyses, e.g., nucleic acid separations, protein sizing
separations, and the like are now routinely performed in
microfluidic systems, e.g., the Agilent 2100 Bioanalyzer and
Caliper LabChip.RTM. systems. Similarly, high throughput analytical
operations, e.g., pharmaceutical screening, high throughput genetic
analysis, and the like, are also being transitioned from multi-well
formats into microfluidic formats, such as the Caliper HTS sipper
chip systems. These microfluidic systems have allowed for increases
in throughput while requiring substantially smaller volumes of
reagents, smaller equipment footprint, and having more
reproducible, automatable, integratable operations.
[0003] As with any advancing technology however, the
miniaturization of analytical chemistries introduces a number of
additional considerations. For example, in conventional scale
chemical or biochemical analyses, problems associated with
interaction between reagents and reaction vessels are kept to a
minimum by virtue of the overwhelming volume of reagents used.
Similarly, the nature of the reaction vessels used in conventional
technologies, while illustrating the advantages of microfluidic
systems, also obviate some of the potential problems of
microfluidic systems. For example, because these reaction vessels
are typically configured as discrete wells or test tubes, there is
little or no issue of interaction between discrete reactions that
are being analyzed. Similarly, the open-top nature of these vessels
allows the evolution of other interfering components, which is not
reasonably practicable in sealed microfluidic channels.
[0004] In enclosed microfluidic systems, however, the channel
surface to volume ratio is substantially increased over
conventional technologies, increasing the effects that those
surfaces have on the contents of those channels. Further, because
of their enclosed nature, one cannot readily access and control the
reactions as they progress through the system. In addition, the
sealed nature of these systems can result in the accumulation of
evolved gasses from the fluid reagents of a system, where such
gases would dissipate into the atmosphere in conventional assay
formats.
[0005] A number of stop-gap measures have been employed in attempts
to address some of these potential problems of microfluidic
systems. For example, U.S. Pat. No. 5,880,071 describes methods of
reducing effects of electrokinetic biasing of reagents within
electrically driven microfluidic channel systems. Similarly, U.S.
Pat. No. 6,043,080 to Lipshutz et al., describes the use of gas
venting membranes within a miniature chamber, to permit degassing
of fluids within a miniature fluidic environment.
SUMMARY OF THE INVENTION
[0006] The present invention is generally directed to methods of
controlling environmental conditions within a fluidic system, where
such environmental conditions can affect the operation of the
system in its desired function, and fluidic channels, devices and
systems that are used in practicing these methods. Such methods are
generally directed to environmental control fluids, the movement of
such fluids through these systems, and the interaction of these
fluids with other components of the system, e.g., other fluids or
solid components of the system.
[0007] In a first aspect, the present invention is directed to a
method of using an environmental control reagent to maintain
optimal conditions within a microfluidic device. The method
comprises introducing a volume of a first fluid into a channel
segment of a microfluidic device, where the first fluid region
comprises an environmental control reagent. A volume of a second
fluid is flowed into the channel segment of the microfluidic device
immediately before or after the step of flowing the volume of the
first fluid through the channel segment.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 is a schematic illustration of serial fluid plugs in
a fluidic channel, in accordance with certain aspects of the
invention.
[0009] FIG. 2 is a schematic illustration of an exemplary
microfluidic device structure.
[0010] FIG. 3 is a schematic representation of a microfluidic assay
device and reagent source used in conjunction with high-throughput
applications of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0011] I. General
[0012] The present invention generally provides methods for
optimizing the operation of microscale channel based systems
through the use of environmental or operation control reagents
within the fluids that are being transported through the capillary
channels. In optionally preferred aspects of the invention, the
environmental or operation control reagents are transported in
fluid regions that are different from fluid regions that contain
the reagents of interest for a given analysis, although such
reagents may optionally be disposed within the fluid regions that
contain the reagents of interest.
[0013] As used herein, the phrase "environmental control reagent"
or "operation control reagent" refers to a reagent that typically
is not involved directly in the reaction of interest, but instead
modifies, controls or provides an indication of the state of the
environment within a microscale channel in which a reaction of
interest is taking place, so as to control that environment or
provide the user with information as to the state of that
environment such that external controls may be applied. Some
specific and preferred examples of environmental controls include
modifying the surface characteristics of the microscale channel,
adjusting the viscosity and/or the channel flow resistance, and
reducing the potential for gas evolution within the channel.
Operation control reagents that are used as indicators or
diagnostic reagents typically include, e.g., pH indicators, redox
indicators, conductivity indicators, and any of a variety of dyes
or labels that indicate the presence or absence of particular
species, e.g., proteins, nucleic acids, etc. Because the
environmental control reagents are flowed through the microscale
channels, either as a constant stream, or in periodic pulses, they
continually modify and control the environment within those
channels or provide constant indicators as to the state of that
environment. This is a particularly useful function where one or
more environmental conditions within a microscale channel can
change over time or deteriorate with respect to the performance of
the reaction of interest.
[0014] The methods, devices and systems described herein are
particularly useful in performing serially oriented microfluidic
analyses, e.g., where analytes are separately and serially
introduced into a microscale channel. The serial processing is also
multiplexed using multiple separate channels, e.g., where a large
number of different analytes are subjected to analysis by serially
introducing each into a separate one of multiple parallel channels.
In such serially processed analyses, it is often desirable,
although not always necessary, to space serially introduced
analytes from each other. This is primarily to provide an ease of
identification of the different analyte regions within a channel,
as well as preventing intermixing of analytes and its potential
deleterious effects on the analysis being carried out. These spacer
fluid regions are ideal vehicles for the environmental control
reagents described herein. In particular, conditions for a given
analytical reaction can be optimized without having to account for
the presence of the environmental control reagent within the
reaction fluid region. Alternatively, a wide variety of different
analytes can be tested without having to mix them with these
environmental control reagents.
[0015] FIG. 1 is a schematic illustration of a serial process
analysis that includes multiple reaction fluid regions interspersed
with spacer fluid regions that include environmental control
reagents. Specifically, reaction fluid regions 104, 108 and 112 are
flowed through a microscale channel segment 102. Spacer fluid
regions 106, 110 and 114, which include the environmental control
reagents, are interspersed between the reaction fluid regions. The
spacer fluid regions 106, 110 and 114 include the environmental
control reagents as described herein. Although the reaction fluid
regions optionally include the environmental control reagents, in
preferred aspects the environmental control reagents are primarily
contained within the spacer fluid regions. This allows the use of a
single source of environmental control reagents, e.g., in a spacer
fluid reservoir, rather than requiring mixing of each of the
different reaction fluids with those reagents. Additionally, to the
extent environmental control reagents might have any effect on the
reactions that are being carried out in the reaction fluid regions,
they are kept substantially separated. Further, as each spacer
fluid region passes through the microscale channel segment, it
performs the particular function or functions for which the reagent
is intended. In this manner, the spacer plugs perform somewhat of a
"housekeeping" function in the microfluidic channel systems.
[0016] In certain preferred aspects, the environmental control
reagent is flowed through the channel segment immediately following
each region of reaction fluid, e.g., such that the fluid region
containing the environmental control reagent(s) are interspersed
among reaction regions. In certain aspects, the environmental
control reagent region is adjacent to and abutting the reaction
fluid region, to optimize the environmental control function that
those reagents perform. However, in those instances where
environmental control functions are not necessary on as routine a
basis, the period between environmental control reagent additions
can be increased, e.g., after 2, 5, 10 or even 20 or more reaction
fluid regions, and their optionally associated spacer fluid
regions, have passed through the particular channel segment.
Conversely, in some cases, an environmental control reagent may be
transported through a channel only once or a few times for the
lifetime use of a given channel structure, and may increase the
useful lifetime of a channel network.
[0017] II. Surface Modifying Reagents
[0018] As noted above, one exemplary environmental control reagent
type is a channel surface modifying reagent. In microscale fluidic
systems, the nature of the surface of the microscale channel
through which materials are being transported can have a
significant effect on the operations that are being performed. For
example, surfaces can have properties, e.g., surface charges,
hydrophobicity, etc., which promotes the sticking of proteins,
cells or other reaction materials to those surfaces. This
accumulation of material can subsequently interfere with material
flowing or other aspects of a reaction of interest. Similarly, in
electrically driven microscale channel systems, the existence of
charged functional groups on the channel surface can give rise to
electrokinetic movement of fluids within channels, where such
movement may be more or less desired.
[0019] Accordingly, in many microscale fluidic systems it is
desirable to treat the surfaces of the channels to mask unfavorable
characteristics or provide or accentuate favorable characteristics.
Previously, such surface treatments have focused upon pretreating
the channel surfaces through coating processes that involved
complex chemical treatments to covalently attach chemical modifying
agents to those channel surfaces. In certain applications, e.g.,
where a channel is used a single time, dynamic coating materials
have been described, where a solution of the surface modifying
agent is disposed within the channel in order to perform the
analysis of interest. While these dynamic coatings are useful in a
variety of applications, e.g., where the entire channel segment
where the analysis is being performed is filled with the dynamic
coating material, their non-permanent nature can be a significant
drawback to their use in a number of other applications, e.g.,
applications where such materials can adversely affect the reaction
of interest, as the channel surface properties will change over
time as the dynamic coatings are washed from the channel
surfaces.
[0020] It is these latter situations that are particularly
advantageously addressed by the methods of the present invention.
Specifically, in applications where one is introducing reaction
fluid regions into a microscale channel, surface modifying reagents
can be introduced into the channel in a separate fluid region that
is flowed through the channel before and/or after the reaction
fluid region. This can be repeated each time a new reaction fluid
region is introduced into the microscale channel segment or at
pre-selected intervals, e.g., regularly spaced intervals. As these
spacer fluid regions or plugs flow through the channel, they
continually coat or re-coat the surface of the channels for a
following reaction fluid region.
[0021] A wide variety of different materials are useful as surface
modification reagents in accordance with the present invention. For
example, as noted above, dynamic coatings that typically comprise
surface adsorbing polymer solutions are used as the surface
modifying reagent. Examples of such polymer solutions include
linear, e.g., non-crosslinked cellulose polymers, agarose polymers,
acrylic polymers, e.g., polyacrylamides, and the like. Particularly
preferred polymers include linear polyacrylamide polymers, and more
particularly linear polydimethylacrylamide polymers (PDMA) and
copolymers of these, e.g., PDMA co-acrylic acid polymers as
described in U.S. Pat. No. 5,948,227, which is incorporated herein
by reference in its entirety for all purposes, monovalent or
bivalent compounds that interact with the surface and present
different environments to the fluids within the microchannel, e.g.,
charged groups, hydrophobic moieties, affinity binding moieties, or
the like, for use in chromatographic analyses, and the like. A
variety of these latter reagents have been described in detail in
the art, and could be readily employed in the present
invention.
[0022] In accordance with this aspect of the invention and with
reference to FIG. 1, the surface modifying reagent is provided in a
first fluid region that is transported through a particular
microscale channel, whereupon the reagent within the first fluid
region modifies the surface of the channel. A second fluid region
is preferably introduced into the channel following the first fluid
region. As the channel surfaces have been previously modified, the
influence of those channel surfaces on the reaction is controlled
as desired, e.g., minimized, reduced, increased, or otherwise
altered for desired effect. In preferred aspects, an additional
first fluid region, e.g., containing the surface modifying reagent,
is then transported through the channel after the reaction fluid
region, to re-coat the channel surfaces. This ensures a consistent
level of environmental control among different, serially introduced
reaction fluid mixtures.
[0023] In a closely related aspect, the environmental control
reagents of the invention can be used to counteract build-up of
other reagents within a microscale channel by performing cleaning
functions within the channel. For example, as noted above,
microscale channels, and particularly uncoated microscale channels
are very susceptible to deposition and accumulation of material
within a flowing system, e.g., as a result of the surface charge or
hydrophobicity of the channel surface. Such accumulation can affect
the continued operation of a microfluidic system by affecting the
concentration of reagents, interfering with detection techniques,
etc. Accordingly, in accordance with the present invention,
environmental control reagents that are flowed through the channels
of the device include "cleaning" agents to remove any accumulated
material from channel walls. In its simplest form, such cleaning
agents include acids (e.g., HCl), bases (e.g., NaOH), detergents,
high salt solutions (NaCl, NH.sub.4SO.sub.4, and the like),
zwitterionic solutions, e.g., amino acid solutions, nondetergent
sulfobetaine (NDSB), and others. Acid or base solutions are
typically used at concentrations of greater than 1 mM and
preferably greater than about 10 mM, and in some cases greater than
50 mM. In such cases, the environmental control reagents may be
separated from sample material containing fluids by a strongly
buffered spacer fluid region, e.g., to avoid damaging the sample
material. Salt concentrations will generally vary depending upon
the nature of the cleaning operation to be carried out and may be
in excess of 10 mM and often in excess of 20 or even 50 mM.
Similarly, in the case of detergents, a variety of detergents are
commercially available and may be employed as desired as an
environmental control reagent.
[0024] III. Viscosity/Flow Resistance Adjusting Reagents
[0025] Environmental control, as used in conjunction with the
present invention may also include control of the overall or
average fluidic characteristics of a microscale channel or channel
network. For example, in pressure driven fluidic systems, e.g.,
systems where fluid flow is controlled by application of pressure
differentials across channels, the rate of flow within channels is
dictated, at least in part, by the level of flow resistance within
a particular channel. Flow resistance of a channel can be
manipulated by adjusting the structural characteristics of the
channel, e.g., its length, width and or depth. However, such
manipulations are typically carried out at the time of
manufacturing of the channel system and are not readily
altered.
[0026] Flow resistance can also be altered by altering the
characteristics of the fluid flowing through the channels. In
particular, by adjusting the viscosity of fluids flowing through
channels, one can alter the overall flow resistance of those
fluids. In accordance with the present invention, certain fluid
regions, e.g., spacer fluid regions, can be viscosity adjusted to
achieve an overall change in flow resistance through the channel.
Implicit in this description is the optional situation where
certain fluid regions are not adjusted for viscosity. This can
result from an inability to practically adjust viscosity in some of
the fluids, due to their numbers, etc., or can be a result of
negative interactions between the viscosity adjusting reagents and
those other fluids, e.g., reaction components.
[0027] A variety of viscosity adjusting reagents may be used in
accordance with the present invention, including polymeric
reagents, e.g., cellulose, agarose, gelatin, polyacrylamides, i.e.,
PDMA and co-polymers thereof, PEGs and other polyalcohols, Ficoll,
hydrogels, and the like.
[0028] IV. Reagents for Controlling Gas Evolution
[0029] Another environmental characteristic that can pose potential
problems in microfluidic channel systems, and for which the present
invention is particularly suited is the variation in dissolved
gases within fluids flowing through the channels of the system. In
particular, where fluids in microfluidic systems have the potential
to evolve dissolved gases, such gases can create substantial
problems in microscale channels, including blocking or otherwise
restricting flows in channel networks, which can substantially
disrupt the efficient operation of those systems. In analytical
reactions, the potential for gas evolution is increased where, as
in many bioanalytical operations, temperatures are maintained at
elevated levels to optimize assay conditions. Similarly, many
microfluidic operations involve the use of pressure gradients to
manipulate fluids within microscale channel networks. Substantial
changes in pressures can lead to outgassing within the channel
system. For example, often fluid flow is driven by an applied
vacuum, where the pressure drop across the channel network can
result in substantial degassing of fluids within the channel
networks, where those fluids are sufficiently saturated.
[0030] In accordance with the present invention, an environmental
control reagent comprises a fluid reagent that is capable of
controlling dissolved gas levels within the channel system at
levels that do not result in gas evolution within the channels
under the conditions of operation. In the simplest aspect, the
environmental control reagent in this context is a fluid reagent,
e.g., buffer, water, etc., that has a dissolved gas level that is
far below the level where gas evolution would be expected in the
operation that is being carried out, e.g., at the temperatures and
pressures or vacuums involved within the microscale channel
systems. Typically, this gas control fluid also has a dissolved gas
level that is sufficiently below that of most, if not all of the
other fluids that are used in the particular analytical operation,
e.g., the reaction fluids, spacer fluids, etc. such that when all
of the fluids are mixed, the resulting solution will not evolve gas
under the operating conditions of the particular operation.
[0031] Because the gas control fluid has such a low level of
dissolved gas, it can serve to scavenge excess dissolved gases from
the other fluid regions within the channel networks, e.g., the
reaction fluid regions, where a degassing operation may not have
been reasonably practicable and as a result may have dissolved gas
levels that could result in gas evolution within the microscale
channel system. In particular, for many operations, e.g., high
throughput analytical operations, it is not reasonably practicable
to de-gas all of the different sample materials that one is
analyzing. Further, in many cases, sample materials are subjected
to numerous in-process, but out-of-channel manipulations, e.g.,
dilutions, mixing, etc. which would effectively negate any attempts
at degassing. By maintaining certain fluids within a channel system
at dissolved gas levels that are well below saturation, one can
balance the effects of higher gas concentrations in other fluids
used in the operation. As used herein, the term "saturation" refers
to the gas saturation point of a given fluid under the then current
conditions within a microscale channel. As a result, the saturation
point, or gas solubility, of a particular fluid at one temperature
and pressure will be different from the saturation point of the
fluid at another temperature and/or pressure.
[0032] The reduced level of dissolved gas within the gas control
reagents, as described herein, is generally dependent upon the
particular operation that is to be carried out, rather than being
an absolute characteristic. In particular, where a desired
operation is to be carried out within a microchannel structure at
lower temperatures, higher absolute concentrations or levels of
dissolved gas can be tolerated without evolution. Conversely, where
a particular operation is carried out at higher temperatures, lower
dissolved gas concentrations are tolerated. Similarly, where
negative pressures are applied to fluids within a channel, e.g., as
compared to ambient pressure of the fluids prior to their
introduction into the channel system, it is generally required that
such fluids have lower dissolved gas concentrations in order to
avoid outgassing within the channels. Conversely, positively
pressurized channels are generally capable of supporting fluids
with higher dissolved gas concentrations.
[0033] From the particular conditions of a given operation, one can
readily determine the level of acceptable dissolved gas, in order
to avoid any problems associated with gas evolution within the
channels, e.g., bubble formation. For example, where one knows the
temperature at which the fluid is maintained within the channel
system, the amount of dissolved gas in some of the fluid reagents,
and the amount of applied vacuum in a given channel, one can
determine the acceptable level of dissolved gas within a spacer
fluid, as well as the relative amount of that spacer fluid needed,
to counter any potential of outgassing within the channel.
Specifically, one can provide a sufficient amount of a spacer fluid
within a channel, where that spacer fluid has a sufficiently low
level of dissolved gas, such that any excessive gas concentrations
of any of the remaining fluid reagents is absorbable by the spacer
fluids without outgassing under the conditions of the
operation.
[0034] By way of example, where a microfluidic channel network is
operated at -2 psi vacuum to cause fluid flow, while the device and
all of the reagents are at room temperature, and where the
environmental control reagent makes up 40% of the overall fluid
volume within the channels of the device, then the environmental
control reagent must be at or below 75% saturation with air at room
temperature and 1 atmosphere pressure.
[0035] Typically, the gas control reagents are only required to be
at a saturation level that is at or below that necessary to prevent
outgassing of a given system under that system's operating
conditions. In accordance with this principle, the degassing fluid
need only have somewhat less. However, in general, this results in
the gas control reagents having a dissolved gas concentration that
is less than 90% of the saturation concentration for any portion of
the operation that is to be carried out within the microscale
channel system. In preferred aspects, the gas control reagent has a
dissolved gas concentration that is less than 80%, more preferably,
less than 60%, more preferably, less than 50%, and often less than
40%, 30% or even 20% of the saturation level of dissolved gas in
any portion of the operation being carried out.
[0036] Although described above in terms of the use of a degassing
reagent or fluid within a microfluidic channel system, it is also
an aspect of the invention to operate microfluidic systems under
conditions that prevent such outgassing, e.g., using any or all
fluids within the channel network to prevent outgassing. In
particular, as noted above, the conditions which prevent outgassing
will generally depend upon other conditions of the system, e.g.,
the applied pressure, the gas saturation level of the fluids within
the channels and the gas saturation level of the fluids prior to
entering the channel. Gas saturation is highly temperature
dependent, e.g., colder fluids can dissolve larger amounts of
oxygen than warmer fluids. Similarly, fluids maintained at a lower
pressure will evolve more gas than fluids kept at higher pressure.
Accordingly, by adjusting one or more of the temperatures of the
fluids before and after entering a microfluidic channel network,
and/or the pressure or vacuum applied to a system, one can ensure
that the system operates under non-out-gassing conditions.
[0037] In particularly preferred aspects, the applied pressure is
determined by the desired flow rate through the system, e.g.,
resulting from desired throughput or reaction times. Accordingly,
prevention of outgassing is typically a matter of adjusting one or
more of the temperatures of the fluids prior to entering the
channels and after entering the device. By way of example, a fluid
that is maintained at a first temperature, but which is heated upon
entering the channel network poses a substantial risk of bubble
generation within the channels. This is particularly true where the
flow of fluids in the channels is driven by vacuum. Accordingly, to
remedy this issue, prior to drawing fluids into the channel
network, one can (1) maintain all of the fluids at a temperature
that is at or above the temperature of the channel network; (2)
elevate the temperature of some portion of fluids (e.g.,
environmental control/degassing fluids) above the temperature of
the channel network; or (3) cool the channel network to a
temperature below that of the outside fluids. Finally, one could
also perform any of these adjustments in conjunction with changes
in the level of applied pressure to the channel system. As channel
temperatures are often optimized for the particular analysis, it is
generally preferable that most of the temperature adjustments be
made to the fluids prior to their entering the channel networks.
This is typically a simple matter of providing a heating element to
the sources of these fluids, e.g., multiwell plates, reagent
troughs, or the like. Again, as described above, relative
temperatures of fluids inside and outside the channel networks are
dependent upon the nature of the overall conditions. Typically,
however, fluids are maintained at least 1.degree. C. over the
temperature of the microscale channel network (also referred to as
the chip temperature), and preferably, more than 5.degree. C. This
is particularly the case where only a portion of the fluids to be
introduced into the chip are provided outside the chip at elevated
temperatures. In some cases higher temperature differentials are
desirable and may be 10, 20, 30.degree. C. or more, e.g., the
temperature of the fluids prior to entering the channel network is
10, 20 or 30.degree. C. higher than the chip temperature.
Alternatively or additionally, one can adjust the pressures to
which the fluids are subjected so as to prevent bubble formation
within a microchannel. For example, in one aspect, one can subject
fluids to a low pressure environment, e.g., below ambient, by
applying a vacuum to the fluids to degas those fluids prior to
introducing them into the channels of the system. Alternatively, or
additionally, one can maintain the pressures within the channels at
levels that prevent such degassing, e.g., above ambient or the
pressure at which the fluids were kept prior to introduction into
the channels.
[0038] In preferred aspects, because fluids are often flowed
through microchannels under an applied vacuum, the latter
alternative is not optimally applicable in all situations. As such,
where pressure adjustments are used to prevent degassing within the
microchannel, it is typically applied as a preloading step, e.g.,
fluids are subjected to vacuum prior to loading into a channel.
[0039] In particularly preferred aspects, the fluids to be
introduced are subjected to either one or both of an elevated
temperature or reduced pressure environment immediately prior to
introducing those fluids into the channels, to prevent
re-equilibration of the fluids at atmospheric temperatures, and so
as not to necessitate complex sealed bottle systems. As used
herein, "immediately prior to introduction" means 5 minutes or less
before fluid introduction, preferably 1 minute or less, and often
30 seconds or less. In certain systems, a trough of fluids is
continuously recirculated and subjected to elevated temperatures.
In the case of subjecting fluids to reduced pressure atmospheres, a
variety of different vacuum degassing methods may be employed,
including bulk degassing of fluids, e.g., subjecting larger volumes
of fluid to negative pressures. However, in the systems described
herein, in-line degassing systems are generally preferred. One
example of an in-line degassing system, available from Agilent
Technologies, applies a negative pressure to one side of a Gore-Tex
fabric membrane while the fluid to be degassed is flowed across the
other side of the membrane. The system allows gas to pass from the
fluid, through the membrane as a result of the applied vacuum,
resulting in effective degassing of the fluid in a flowing format.
The degassed fluid is then flowed into a trough for sampling, or is
delivered directly to a port of a microfluidic device for
introduction into the channels of the device.
[0040] Although the preferred gas control reagents comprise fluids
having sufficiently low dissolved gas concentrations, e.g.,
buffers, water etc., it will be appreciated that gas absorbing
additives may also be used in conjunction with this aspect of the
present invention, in order to reduce the potential for outgassing
during a given operation. For example, in certain embodiments, it
may be useful to employ liquids that have very high oxygen
saturation levels, i.e., fluorocarbons and perfluorocarbons.
[0041] V. Other Operational Control Reagents
[0042] In addition to the environmental control reagents described
above, intermediate fluid slugs in microfluidic channels can be put
to a variety of other useful functions in microfluidics based
analyses. For example, spacer fluid slugs optionally incorporate
signaling components, e.g., dyes, labels, etc., so as to provide an
indicator component of the spacer slugs versus the sample slugs.
Such indicators may be varied in terms of the nature of the label,
e.g., in the case of fluorescent labels, via its wavelength of
excitation, emission, intensity or the like, to provide the ability
to distinguish between spacer slugs at different points in an
operation. By way of example, a spacer slug early in an assay run
may have a first signaling component, e.g., fluorescing at a first
wavelength or combination of wavelengths, whereas later spacer
slugs fluoresce at a different wavelength or combination of
wavelengths. Depending upon the nature of the spacer slug's label,
one can determine where the system is in a given operation, in
order to identify intermediate steps in an operation, or match
analysis data with exogenously introduced reagent slugs, e.g.,
allowing identification of the particular reagent slug. Such
different labels can be provided by sampling spacer fluids from
different sources at different time points in an operation, or by
constantly adjusting the signal component make-up of a single
source of spacer fluid. For example, a trough of spacer fluid may
be slowly, but constantly supplemented with a new labeled signaling
component, so as to produce an increasing level of signal in later
sampled spacer fluid slugs.
[0043] Spacer slugs containing signaling components may also be
used as calibrators for detection systems used in conjunction with
the devices and methods described herein. In particular, a known
concentration of signaling component can be used to set detection
system so as to optimally detect assay results.
[0044] In addition to the foregoing, signaling components of spacer
slugs can be used as indicators or diagnostics of other
environmental conditions within a microscale channel system. For
example, temperature sensitive signaling components may be used to
monitor temperature within such systems over the course of an assay
run, while pH sensitive signaling components may be used to
indicate intrachannel pH or changes therein. Additionally, ion
specific indicators, or generic conductivity indicators may be
used. A variety of temperature sensitive signaling components may
be used in conjunction with this aspect of the invention, including
molecular beacons, self hybridizing nucleic acid sequences that
become fluorescent when heated above their melting temperatures.
Similarly, pH indicating labels or dyes are widely available from
commercial sources, including, e.g., Molecular Probes, Inc.
(Eugene, Oreg.). Dyes or labels can also be provided to indicate
the level of macromolecular buildup within channels, which buildup
might affect the functions of the channels, or their usefulness in
a given analysis. Such dyes include protein indicators, nucleic
acid indicators, and the like.
[0045] VI. Microscale Channels and Systems
[0046] As noted repeatedly above, the present invention is most
useful in the context of analytical operations that are carried out
within a sealed microscale channel environment. In its simplest
form, such an environment includes a simple channel, e.g., a
capillary, tube or other enclosed conduit through which fluid
materials are flowed. However, in preferred aspects, the operations
in question are carried out within more complex networks of
microscale fluid channels, e.g., in microfluidic devices.
Typically, such devices include at least two different microscale
channels disposed within the same single body structure. Often, the
at least two microchannels will be in fluid communication with each
other, e.g., at a channel junction, to form an integrated channel
network. In general, microfluidic devices incorporating complex
channel geometries have been previously described in, e.g., U.S.
Pat. Nos. 5,869,004, 5,942,443, 5,976,336, 6,042,709 and 6,068,752,
each of which is incorporated herein by reference in its entirety
for all purposes. As used herein, the term microchannel typically
refers to a channel conduit that has at least one cross-sectional
dimension between 0.1 and 500 .mu.m. Preferably, at least one
cross-sectional dimension of a microchannel is between about 1 and
about 100 .mu.m.
[0047] While microfluidic devices may be fabricated as an aggregate
of different parts, e.g., capillaries and chambers, pieced together
in a desired orientation, in preferred aspects, such devices are
fabricated in a monolithic format, integrated in solid substrates.
In particular, microscale channels and channel networks are
typically fabricated as grooves into a surface of at least one
planar substrate layer. The first substrate layer is then overlaid
with a second substrate layer, which is bonded to the first, to
seal and enclose the grooves as microscale channels. Reservoirs or
access ports are optionally provided in one or both of the
substrate layers to provide access to the channels from the outside
world. Additional substrate layers are optionally added to increase
to the complexity of channel networks that may be produced.
Similarly, individual channel networks may be duplicated within one
or more different body structures, in order to multiplex
operations, and gaining the consequent improvements in throughput.
FIG. 2 provides a schematic illustration of the assembly of a
layered microfluidic device. As shown, the device 10 includes a
lower planar substrate 12 having a plurality of grooves fabricated
into its surface. An upper substrate layer 18 is also provided that
includes a plurality of apertures disposed through it. The
apertures are positioned so as to be in communication with the
grooves when the upper layer is placed upon and bonded to the lower
substrate. This bonding also seals the grooves as enclosed channels
or conduits. Although illustrated as grooves on the lower substrate
and apertures through the upper substrate, it will be appreciated
that grooves and apertures may be disposed in either and/or both
substrates depending upon the desired nature of the finished
microfluidic device.
[0048] Methods for manufacturing microfluidic devices have been
previously described, and include techniques commonly employed in
the integrated circuit industries, e.g., photolithography and wet
chemical etching, for silica based solid substrates, as well as
other well known microfabrication techniques for other materials,
e.g., injection molding and embossing techniques for polymer-based
materials (see, e.g., U.S. Pat. No. 5,885,470).
[0049] Generally, such devices are mounted on an instrument that
includes fluid transport systems, as well as detection systems,
whereby the instrument interfaces with the microfluidic device to
control fluid movement and detect assay results within the channels
of the microfluidic device. Such instruments are exemplified by,
e.g., the Agilent Technologies 2100 Bioanalyzer and the Caliper
Technologies HTS "Sipper" platform, as described at
www.calipertech.com and www.Agilent.com, the contents of which are
hereby incorporated herein by reference in their entirety for all
purposes. Microfluidic devices, methods and systems that include
serially introduced fluidic regions, e.g., as described in the
preferred embodiments of the invention, are described in
substantial detail in U.S. Pat. Nos. 5,942,443, and 6,042,709, each
of which is incorporated herein by reference in its entirety for
all purposes.
[0050] The devices and methods of the invention may be employed in
conjunction with appropriate instrumentation depending upon the
nature of the analysis that is to be performed. For example, for
lower throughput operations, microfluidic devices are readily
configured for operation on commercially available
controller/detector instrument, e.g., an Agilent 2100 Bioanalyzer
that is equipped with at least one pressure/vacuum source.
Similarly, higher throughput operations are readily configured to
operate on sipper systems that are available from Caliper
Technologies Corp. Such systems are described in detail at
www.agilent.com and www.calipertech.com and in U.S. Pat. Nos.
5,955,028, 6,042,709 and 6,071,478, each of which is incorporated
herein by reference in its entirety for all purposes, as well as
the patents described elsewhere herein, and incorporated herein by
reference.
[0051] VII. Examples
[0052] The degassing functions of the present invention were
modeled and applied in a high-throughput screening system that
incorporates a microfluidic channel network. A simplified schematic
illustration of the microfluidic device and overall system is shown
in FIG. 3. As shown, the microfluidic device 300 includes a planar
body structure 302 that includes a channel network disposed within
its interior. The channel network includes a main analysis channel
304 that is coupled at one end to an external capillary element
306, via inlet 308. At the other end, the main channel 304 is
fluidly connected to reservoir/port 310. Two side channels 312 and
314 intersect and are in fluid communication with the main channel
304. These channels provide a connection between the main channel
304 and reagent reservoirs 316 and 318, respectively. In the
examples described below, sample material is sampled into the main
analysis channel through the external capillary 306 by dipping the
open end of the capillary into a source of sample material 320 and
applying a vacuum at reservoir/port 310. The applied vacuum draws a
slug of sample material into the capillary element 306 and moves it
into the analysis channel 304. In the system shown, a spacer fluid
is introduced after the sample material slug, in order to space the
sample material from subsequent sample materials. The spacer buffer
is sampled into the system the same way that the sample is drawn
in. Specifically, the capillary element is placed into contact with
the trough 322 of spacer buffer and a slug of spacer fluid is drawn
into the system. Within main channel 304, additional reagents
needed for a given analysis are brought into the main channel 304
from the side channels 312 and 314. Movement of reagents into
channel 304 from these side channels is driven by the same vacuum
used to draw materials in through the capillary element. In the
context of the present example, the spacer fluid constituted the
degassing fluid.
[0053] Degassing parameters were calculated for a system having the
attributes described with reference to FIG. 3. In particular, a
number of physical and temporal parameters of the operation of a
microfluidic device are dictated by the particular analysis to be
carried out therein. Those parameters were then used to calculate
the maximal allowable level of oxygen within the spacer fluid in
order for that fluid to function as a degassing reagent. In order
to achieve this oxygen level, therefore, a minimum spacer fluid
trough temperature was calculated, e.g., to provide spacer fluid at
an acceptable oxygen level. By providing the spacer fluid below
maximal oxygen levels, substantial reductions in bubble formation
and channel blockage have been observed. Exemplary calculations are
provided below.
[0054] In one exemplary analysis, sample compounds are sipped for 2
seconds, while spacer fluids are sipped for 1 second. Transit time
between the sample well 320 and the spacer fluid trough 322 is 1.5
seconds. For the particular example chip/channel configuration,
flow into main channel 304 from the side channels is 50% of the
total flow, e.g., 25% from each side channel. The temperature of
the sample material is assumed to be room temperature or 22.degree.
C., while the device temperature is elevated to 28.degree. C. This
elevated temperature is generally desirable to accelerate analysis
chemistries within the device. Finally, a vacuum of -0.3 psi is
applied to reservoir/port 310 to drive fluid flow through the
channels of the device. These parameters were then used to
calculate the maximal level of oxygen within the spacer fluid in
order to avoid any degassing or bubble formation within the
channels of the device, e.g., under the temperature and pressure
conditions applied. In carrying out the calculations, two alternate
scenarios were assumed. The first case is where a hanging droplet
of fluid at the end of the capillary element does not equilibrate
with air during the transit time from the sample to the trough. The
second case assumes that the droplet becomes fully equilibrated
with air during transit. Given that the radius of the water droplet
is 0.018 cm and the diffusion constant for oxygen in water is
2.times.10.sup.-5 cm.sup.2/s, giving a diffusion time of 8.1
seconds, the droplet should not equilibrate during a typical
transit time, e.g., 1.5 seconds. The calculations are set forth
below:
1 Input Parameters Parameter ID Value Sample plate sip time (s) PST
2 Trough sip time (s) TST 1 Sipper transit time (s) STT 1.5 Side
channel flow (fraction) SAF 0.5 Sample plate temperature (.degree.
C.) PT 22 Chip temperature (.degree. C.) CT 28 Applied vacuum (psi)
V 0.3
[0055]
2 Calculated Parameters Parameter ID Case 1 Case 2 Sample plate
flow (%) FP 29.2 41.7 Trough flow (fraction) FT 20.8 8.3 [O.sub.2]
in plate OP 4.17 .times. 10.sup.-3 4.17 .times. 10.sup.-3 [O.sub.2]
in side channel reservoirs OS 3.71 .times. 10.sup.-3 3.71 .times.
10.sup.-3 [O.sub.2] allowable in chip OAC 3.63 .times. 10.sup.-3
3.63 .times. 10.sup.-3 [O.sub.2] allowable in trough OAT 2.7
.times. 10.sup.-3 2.7 .times. 10.sup.-3 Minimum trough temp. MTT 50
94 % Saturation in trough % O2 65 12
[0056] The calculations used to calculate these parameters were as
follows:
FP1=[(PST+STT)/(PST+TST+2*STT)][1-SAF]
FP2=[(PST+2*STT)/(PST+TST+2*STT)][1-SAF]
FT1=[(TST+STT)/(PST+TST+2*STT)][1-SAF]
FT2=[TST/(PST+TST+2*STT)][1-SAF]
[0057] [O.sub.2] in grams of gas/100 grams of water at atmospheric
pressure and temperature T is
[O.sub.2]=13417e-5*(T)-1.301e-8*(T-58.04){circumflex over (
)}3+4.310e-3
OAC=OS*((14.7-V)/14.7)
OAT=(OAC-OP*FP(n)-)S*SAF)/FT(n) where (n) is 1 or 2 depending on
the case.
MTT=(100-6.628e4*(O.sub.2)+1.5e7(O.sub.2){circumflex over (
)}2-1.084e9(O.sub.2){circumflex over (
)}3)/1-5.703e2(O.sub.2)+1.443e5(O.- sub.2){circumflex over (
)}2-2.563e7(O.sub.2){circumflex over ( )}3+3.298e9(O.sub.2))
% O.sub.2=(OAT/OP)*100
[0058] As noted above, when the trough is maintained above the
calculated minimum trough temperature for a given operation,
channel plugs resulting from outgassing within channels is
substantially reduced.
[0059] 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 incorporated by reference. Although the present
invention has been described in some detail by way of illustration
and example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
* * * * *
References