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Claims  |
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What is claimed is:
1. A method of separating macromolecules by capillary electrophoresis,
comprising:
providing a substrate comprising at least a first capillary channel
disposed therein, a surface of the channel having a first surface charge
associated therewith;
filling said capillary channel with a water soluble hydrophilic polymer
solution having a percent charge of from about 0.01% to about 2%, as
calculated by the molar percent of charged monomer subunits to total
monomer utilized in producing the polymer, the charged monomer subunits
consist of monomer subunits having a charge that is the same as the first
surface charge;
introducing a sample containing the macromolecules into one end of the
capillary channel and;
applying a voltage gradient across the length of the capillary channel,
whereby the macromolecules in the sample are separated in the capillary
channel.
2. The method of claim 1, wherein the substrate provided in the providing
step comprises a first surface charge that is negative, and the charged
monomer subunits in the filling step consist of negatively charged monomer
subunits.
3. The method of claim 2, wherein the negatively charged monomer units are
selected from acrylic acid, bisacrylamidoacetic acid,
4,4-Bis(4-hydroxyphenyl)pentanoic acid, 3-butene-1,2,3-tricarboxylic acid,
2-carboxyethylacrylate, itaconic acid, methacrylic acid, 4-vinylbenzoic
acid, sulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid,
2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid,
4-styrenesulfonic acid, 2-sulfoethyl methacrylate,
3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt,
3-sulfopropyl methacrylate, vinylsulfonic acid,
Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.
4. The method of claim 1, wherein the substrate provided in the providing
step is a silica-based substrate.
5. The method of claim 4, wherein the substrate provided in the providing
step comprises a silica substrate, and the polymer in the filling step
comprises polydimethylacrylamide-co-acrylic acid.
6. The method of claim 1, wherein the substrate provided in the providing
step comprises a solid polymeric substrate.
7. The method of claim 6, wherein the solid polymeric substrate is selected
from the group of polydimethylsiloxanes (PDMS), polymethylmethacrylate
(PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone,
polycarbonate, polytetrafluoroethylene.
8. The method of claim 1, wherein the sample contains a plurality of
different nucleic acid sequences.
9. The method of claim 8, wherein the different nucleic acids comprise a
plurality of different fragments of a target nucleic acid sequence.
10. The method of claim 9, wherein the different nucleic acids comprise a
nested set of fragments of a target nucleic acid sequence.
11. The method of claim 10, wherein the each fragment in the nested set of
fragments differs from at least one other fragment in the nested set by
the addition or omission of a single nucleotide at a terminus of the
fragment.
12. The method of claim 1, wherein the capillary channel provided in the
providing step intersects and is fluidly connected with at least a second
capillary channel disposed in the substrate.
13. The method of claim 1, wherein the capillary channel provided in the
providing step intersects and is fluidly connected with at least second
and third capillary channels disposed in the substrate.
14. The method of claim 13, wherein the applying step comprises
simultaneously applying a voltage gradient across each of the first and
second capillary channels, to transport the sample from the second channel
into the first channel and to separate macromolecules in the sample in the
first channel.
15. The method of claim 13, wherein the applying step comprises
simultaneously applying a voltage gradient across each of the first,
second and third capillary channels.
16. The method of claim 1, wherein the polymer in the polymer solution has
a net charge of between about 0.01% and 1%.
17. The method of claim 1, wherein the polymer in the polymer solution has
a net charge of between about 0.01% and 0.5%.
18. The method of claim 1, wherein the polymer in the polymer solution has
a net charge of between about 0.05% and 0.2%.
19. The method of claim 1, wherein the polymer solution comprises a polymer
concentration of between about 0.01% and about 20% (w/v).
20. The method of claim 1, wherein the polymer solution comprises a polymer
concentration of between about 0.1% and about 10% (w/v).
21. The method of claim 1, wherein the polymer solution has a viscosity of
between about 2 centipoise and about 1000 centipoise.
22. The method of claim 1, wherein the polymer solution has a viscosity in
a range of from about 5 centipoise to about 200 centipoise.
23. The method of claim 1, wherein the polymer solution comprises a
viscosity in a range of from about 10 centipoise to about 100 centipoise.
24. The method of claim 1, wherein the polymer comprises a molecular weight
from about 1 Kd, to about 5,000 Kd.
25. The method of claim 1, wherein the polymer is a polydimethylacrylamide
polymer and the charged monomer is acrylic acid.
26. A method of separating macromolecules by capillary electrophoresis,
comprising:
providing a silica substrate having a capillary channel disposed therein, a
surface of the channel having a negative surface charge associated
therewith;
filling said capillary channel with a water soluble hydrophilic polymer
solution having a net charge of from about 0.1% to about 2%, the charge
being the same as the surface charge;
introducing a sample containing the macromolecules into one end of the
capillary channel; and
applying a voltage gradient across the length of the capillary channel,
whereby the macromolecules in the sample are separated in the capillary
channel.
27. A method of preparing a walled capillary channel for use in separating
macromolecules, comprising:
filling the capillary channel with a silica adsorbing polymer solution,
wherein the polymer has a net charge that is the same as a net charge
associated with interior surfaces of the walled capillary channel.
28. A system for separating macromolecules by capillary electrophoresis,
comprising:
a substrate having at least a first walled capillary channel disposed
therein, the channel having a net surface charge associated with interior
surfaces of the channel;
a solution of silica adsorbing polymer disposed in the capillary channel,
the solution of polymer comprising:
a molecular weight between about 1 Kd and 5,000 Kd;
a net charge of between about 0.01 and 2%, the net charge being the same as
the net surface charge; and
a power source electrically coupled to the first capillary channel for
applying a voltage gradient across the capillary channel.
29. The system of claim 28, wherein the net surface charge associated with
the interior surfaces of the capillary channel is negative.
30. The system of claim 29, wherein the substrate is a silica substrate.
31. The system of claim 30, wherein the substrate is selected from a silica
capillary tube and an etched planar silica substrate.
32. The system of claim 28, wherein the substrate comprises a solid
polymeric substrate.
33. The system of claim 32, wherein the solid polymeric substrate is
selected from the group of polydimethylsiloxanes (PDMS),
polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC),
polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene.
34. The system of claim 28, wherein the substrate further comprises at
least a second walled capillary channel disposed in the substrate, the
second walled capillary channel intersecting and in fluid communication
with the first walled capillary channel.
35. The system of claim 34, wherein the power source is electrically
coupled to each of the fist and second capillary channels, the power
supply simultaneously applying a voltage gradient across a length of each
of the first and second capillary channels.
36. The system of claim 28, wherein the polymer has a net charge between
about 0.01% and about 1%.
37. The system of claim 28, wherein the polymer has a net charge between
about 0.01% and 0.5%.
38. The system of claim 28, wherein the polymer has a net charge between
about 0.05% and 0.2%.
39. The system of claim 28, wherein the polymer solution comprises a
polymer concentration in a range of from about 0.01% to about 20% (w/v).
40. The system of claim 28, wherein the polymer solution comprises a
polymer concentration in a range of from about 0.1% to about 10% (w/v).
41. The system of claim 28, wherein the polymer solution comprises a
viscosity of between about 2 centipoise and about 1000 centipoise.
42. The system of claim 28, wherein the polymer solution comprises a
viscosity in a range of from about 5 centipoise to about 200 centipoise.
43. The system of claim 28, wherein the polymer solution comprises a
viscosity in a range of from about 10 centipoise to about 100 centipoise.
44. The system of claim 28, wherein the polymer is an acrylic polymer and
the charged monomer subunits are selected from acrylic acid,
bisacrylamidoacetic acid, 4,4-Bis(4-hydroxyphenyl)pentanoic acid,
3-butene-1,2,3-tricarboxylic acid, 2-carboxyethylacrylate, itaconic acid,
methacrylic acid, 4-vinylbenzoic acid, sulfonic acid,
2-acrylamido-2-methyl-1-propanesulfonic acid,
2-methyl-2-propene-1-sulfonic acid, 2-propene-1-sulfonic acid,
4-styrenesulfonic acid, 2-sulfoethyl methacrylate,
3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt,
3-sulfopropyl methacrylate, vinylsulfonic acid,
Bis(2-methacryloxyethyl)phosphate, and monoacryloxyethyl phosphate.
45. The system of claim 28, wherein the polymer comprises
polydimethylacrylamide-co-acrylic acid.
46. The system of claim 28, wherein the polymer has a net negative charge.
47. The system of claim 28, wherein the polymer is made by the process of
polymerizing dimethylacrylamide monomers in the presence acrylic acid, the
acrylic acid being present at a concentration of between about 0.01 and 2%
of a total monomer concentration.
48. The system of claim 28, wherein the first net surface charge is capable
of supporting an electroosmotic mobility of a buffer comprising from about
1 mM to about 10 mM sodium borate buffer, at a pH of from about 7 to about
9, disposed in the walled capillary channel, the electroosmotic mobility
being at least about 1.times.10.sup.-5 cm.sup.2 V.sup.-1 s.sup.-1.
49. A system for separating nucleic acids by molecular weight, comprising:
a silica substrate having a walled capillary channel disposed therein, the
channel having a negative charge associated with interior surfaces of the
channel;
a solution of silica adsorbing polymer disposed in the capillary channel,
the solution of polymer comprising:
a molecular weight between about 1 Kd and 5,000 Kd;
a net negative charge of between about 0.01 and 2%; and
a power source for applying a voltage gradient across the capillary
channel.
50. A walled capillary for separating macromolecules by capillary
electrophoresis, comprising:
a capillary channel disposed in a solid substrate, interior surfaces of the
capillary channel having a first net surface charge associated therewith;
and
a solution of silica adsorbing polymer disposed in the capillary channel,
the polymer comprising:
a molecular weight between about 1 Kd and about 5,000 Kd;
a net charge of between about 0.01 and 2%, the net charge being the same as
the first net surface charge. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
Capillary electrophoresis has been established as a highly effective method
for separating macromolecular species in order that they might be further
characterized. Protein and nucleic acid molecules are two major examples
of molecular species that are routinely fractionated and characterized
using capillary electrophoretic systems. These systems have generally
proven effective as a result of the high surface to volume ratio of the
thin capillaries. This high surface to volume ratio allows for much
greater heat dissipation, which in turn, allows application of greater
electrical currents to the capillary thereby resulting in a much more
rapid separation of macromolecules introduced into the system.
In the capillary electrophoretic, size-based separation of biological
macromolecules of interest, e.g., proteins and nucleic acids,
electrophoretic separation is not possible in a free solution. Instead,
such separation requires the presence of a matrix that alters the
electrophoretic mobilities of these molecules based upon their relative
size.
Although early capillary electrophoresis systems utilized solid gel
matrices, e.g., cross-linked polyacrylamides, more recent systems have
employed liquid polymer solutions as a flowable matrix, which permits
adequate separation efficiencies without the drawbacks of cross-linked
capillary systems, i.e., in introducing such matrices to or removing them
from capillary channels.
For example, U.S. Pat. No. 5,126,021 reports a capillary electrophoresis
element which includes a capillary electrophoresis tube containing a low
viscosity uncharged polymer solution, for separating nucleic acids.
U.S. Pat. No. 5,264,101 to Demorest et al. reports the use of a hydrophilic
polymer solution, which is characterized by a molecular weight of 20 to
5,000 Kd, and a charge between 0.01 and 1% as measured by the molar
percent of total monomer subunits to total polymer subunits, where the
charge is opposite to the charge of the surface of the capillary in which
the polymer is used. This opposite charge of the polymer is reported to
result in an interaction between the polymer and the capillary wall to
reduce electroosmotic flow within the capillary.
U.S. Pat. Nos. 5,552,028 and 5,567,292, both to Madabhushi et al., report
the use of a uncharged, water soluble, silica adsorbing polymer in a
capillary electrophoresis system to reduce or eliminate electroosmotic
flow.
Surprisingly, the present inventor has discovered that polymer solutions
can be used in capillary channel systems, which polymers employ a charge
that is the same as that of the internal capillary surface, e.g., positive
or negative. Even more surprisingly, it has been discovered that
electroosmotic flow in capillary channel systems containing such polymer
solutions is maintained the same level or lower than with an uncharged
polymer solution. The present invention provides such polymers, as well as
methods of utilizing these polymers and systems employing such polymers.
SUMMARY OF THE INVENTION
The present invention generally provides novel methods and compositions for
use in the separation of molecular, and particularly macromolecular
species by electrophoretic means.
For example, in an aspect of the present invention is provided a method of
separating macromolecules by capillary electrophoresis. The method
generally comprises providing a substrate which includes at least a first
capillary channel disposed therein, where a surface of the channel has a
first surface charge associated therewith. The capillary channel is filled
with a water soluble hydrophilic polymer solution which includes a percent
charge of from about 0.01% to about 2%, as calculated by the molar percent
of charged monomer subunits to total monomer utilized in producing the
polymer. The charged monomer subunits have a charge that is the same as
the first surface charge. A sample containing macromolecules is introduced
into one end of the capillary channel and a voltage gradient is applied
across the length of the capillary channel, whereby the macromolecules in
the sample are separated in the capillary channel. In preferred aspects,
the surface charge of the capillary channel, as well as the charged
monomer subunits bear a negative charge. In further preferred aspects, the
capillary channel is disposed within a silica substrate.
In a related aspect, the present invention also provides systems and
apparatus for practicing the above methods. In particular, the present
invention provides a system for separating macromolecules by capillary
electrophoresis. The system comprises a substrate having at least a first
walled capillary channel disposed therein, where the channel includes a
net surface charge associated with its interior surfaces. A solution of
silica adsorbing polymer as described above, is disposed in the capillary
channel. The system also includes a power source electrically coupled to
the capillary channel for applying a voltage gradient across the capillary
channel.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 schematically illustrates a silica microscale electrophoresis device
for use in electrophoretic separation of sample component for up to 12
different sample materials, in accordance with the present invention.
FIGS. 2A-2D illustrate the chromatographic separation of DNA standard
samples in a silica microscale integrated channel electrophoresis device
first filled with a neutral polymer solution.
FIGS. 3A-3D illustrate the chromatographic separation of DNA standard
samples in a silica microscale integrated electrophoresis device first
filled with a polymer solution having a negative charge associated with
it.
FIG. 4 illustrates a chromatographic separation as in FIG. 3, but employing
a charged polymer that has a larger average molecular weight and viscosity
than the polymer solution used in generating the chromatogram shown in
FIG. 3.
FIG. 5 illustrates a chromatographic separation as in FIG. 4, except
employing a polymer solution that has a still larger molecular weight and
viscosity than the polymer used in generating the chromatogram shown in
FIG. 4.
FIG. 6 illustrates a channel geometry for a planar polymeric
substrate/microscale channel device used to perform macromolecular
separations in accordance with the present invention.
FIG. 7 illustrates a chromatographic separation of a 100 bp ladder in a
polymethylmethacrylate microfluidic device using a polymer of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of electrophoretically separating
macromolecular species, as well as compositions and systems useful in
carrying out such methods. Specifically, the methods of the present
invention comprise providing a substrate that has at least a first
capillary channel disposed therein. The surface of the channel has a first
surface charge associated therewith, and is filled with a water soluble
surface adsorbing polymer solution that bears a net charge that is similar
to or the same as the charge on the capillary surface, e.g., positive or
negative.
As used herein, the term substrate typically refers to a solid substrate in
which a capillary channel is disposed. Exemplary substrates include silica
based substrates, such as silica, e.g., glass, quartz or the like,
silicon, etc., polymeric substrates, e.g., plastics like
polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane,
polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate,
polytetrafluoroethylene (Teflon.TM.), and a variety of others that are
well known in the art. Substrates may take a variety of shapes or forms,
including tubular substrates, e.g., polymer or fused silica capillaries,
or the like. In preferred aspects, however, the substrate comprises a
planar body structure in which grooves are fabricated to define capillary
channels when overlaid with a cover element, also typically planar in
structure. Examples of such planar capillary systems are described in
commonly assigned copending U.S. application Ser. No. 08/845,754, filed
Apr. 25, 1997 and incorporated herein by reference.
Capillary channels also can be any of a variety of different shapes in
cross-section, including tubular channels, rectangular channels, rhomboid
channels, hemispherical channels or the like, or even more arbitrary
shapes, such as may result from less precise fabrication techniques, e.g.,
laser ablation. Typically, the shape of a capillary channel will vary
depending upon the substrate type used and the method of fabrication. For
example, in typical fused silica capillaries, the capillary channel will
be tubular. In systems employing planar substrates, on the other hand,
channels will typically comprise either a rhomboid, rectangular or
hemispherical cross sectional shape, depending upon the substrate material
and method of fabrication of the channels.
A variety of manufacturing techniques are well known in the art for
producing microfabricated channel systems. For example, where such devices
utilize substrates commonly found in the semiconductor industry,
manufacturing methods regularly employed in those industries are readily
applicable, e.g., photolithography, wet chemical etching, chemical vapor
deposition, sputtering, electroforming, etc. Similarly, methods of
fabricating such devices in polymeric substrates are also readily
available, including injection molding, embossing, laser ablation, LIGA
techniques and the like. Other useful fabrication techniques include
lamination or layering techniques, used to provide intermediate microscale
structures to define elements of a particular microscale device.
Typically, the capillary channels will have an internal cross-sectional
dimension, e.g., width, depth, or diameter, of between about 1 .mu.m and
about 500 .mu.m, with most such channels having a cross-sectional
dimension in the range of from about 10 .mu.m to about 200 .mu.m.
In particularly preferred aspects, planar microfabricated devices employing
multiple integrated microscale capillary channels are used. Briefly, these
planar microscale devices employ an integrated channel network fabricated
into the surface of a planar substrate. A second substrate is overlaid on
the surface of the first to cover and seal the channels, and thereby
define the capillary channels.
One or more analysis channels are provided in the device with additional
channels connecting the analysis channel to multiple different sample
reservoirs. These reservoirs are generally defined by apertures disposed
in the second overlaying substrate, and positioned such that they are in
fluid communication with the channels of the device. A variety of specific
channel geometries are employed to optimize channel layout in terms of
material transport time, channel lengths and substrate use. Examples of
such microscale channel network systems are described in detail in U.S.
application Ser. Nos. 60/060,902, filed Oct. 3, 1997, and incorporated
herein by reference in its entirety. One specific example of a channel
geometry is illustrated in FIG. 1. In operation, sample materials are
placed into one or more of the sample reservoirs 116-138. A first sample
material, e.g., disposed in reservoir 116, is then loaded by
electrokinetically transporting it through channels 140 and 112, and
across the intersection with the separation channel 104, toward load/waste
reservoir 186 through channel 184. Sample is then injected by directing
electrokinetic flow from buffer reservoir 106 through analysis channel 104
to waste reservoir 108, while pulling back the sample in the loading
channels 112:114 at the intersection. While the first sample is being
separated in analysis channel 104, a second sample, e.g., that disposed in
reservoir 118, is preloaded by electrokinetically transporting it into
channels 142 and 112 and toward the load/waste reservoir 184 through
channel 182. After separation of the first sample, the second sample is
then loaded across the intersection with analysis channel 104 by
transporting the material toward load/waste reservoir 186 through channel
184.
The interior surface of the capillary channels typically has a charge
associated with it. For example, in the case of capillary channels
disposed in silica-based substrates, e.g., glass or quartz, the interior
surface of the channel typically includes negatively charged chemical
groups, e.g., silane groups, associated with it. Similarly, polymeric
substrates also typically comprise some level of charged chemical groups
at their surface, although at much lower level than in the case of
silica-based substrates. As used herein, a "charged surface" of a
capillary is typically characterized by its ability to support an
electroosmotic mobility of a fluid or material in the channel. In
particular, channels having charged surfaces as described herein, are
typically capable of supporting an electroosmotic mobility (.mu.EO) of at
least about 1.times.10.sup.-5 cm.sup.2 V.sup.-1 s.sup.-1, for a buffer
when that buffer is in contact with those walls, e.g., disposed within
those channels, e.g., a buffer of from about 1 mM to about 100 mM sodium
borate at a pH of from about 6 to about 9. For the purposes of the present
invention, .mu.EO is defined in terms of a standard buffer of from about 1
mM to about 10 mM sodium borate buffer, at a pH of from about 7 to about
9, for example, 5 mM sodium borate, pH 7. In more common aspects, the
charged surfaces in contact with the fluid are capable of supporting a
.mu.EO under the above conditions, of at least about 2.times.10.sup.-5
cm.sup.2 V.sup.-1 s.sup.-1, preferably, at least about 5.times.10.sup.-5
cm.sup.2 V.sup.-1 s.sup.-1, and in particularly preferred aspects, at
least about 1.times.10.sup.-6 cm.sup.2 V.sup.-1 s.sup.-1.
Different surfaces can also be treated to present differing levels or types
of charged groups. Examples of such surface treatments are described in
detail in copending, commonly assigned U.S. application Ser. No.
08/843,212, filed Apr. 14, 1997, now U.S. Pat. No. 5,885,470, and
incorporated herein by reference in its entirety for all purposes. In
particularly preferred aspects of the present invention, capillary
channels disposed in silica substrates are used, e.g., planar silica
substrates or fused silica capillaries.
In aqueous systems, when charged capillary surfaces are combined with
electric fields necessary for electrophoretic separation, electroosmotic
flow results. For many separations, e.g., protein separations, some
electroosmotic flow is actually desired, in order to ensure a net movement
of all proteins through a capillary channel and past a detector. However,
it is generally desirable to be able to precisely control that level of
flow. In the capillary electrophoretic separation of nucleic acids on the
other hand, it is generally desirable to suppress electroosmotic flow
entirely, to enhance resolution of separation. Further, such charged
surfaces have been implicated in the binding of components of samples,
e.g., proteins, etc., which binding has been blamed for reduced efficiency
of separation.
In accordance with the methods of the present invention, the above
described capillary channel or channels are filled with a solution of a
water-soluble silica-adsorbing polymer. The polymer typically includes a
percent charge of between about 0.01% and 2% that is the same as the
charge that is associated with the interior wall surface of the capillary
channel. By "a charge that is the same as the charge of the interior
surface of the capillary channel" is meant that the polymer includes
charged monomer subunits that are the same charge, e.g., negative or
positive, as the charged chemical groups on the interior surface of the
capillary channel. Thus, where a capillary channel includes negatively
charged groups on the interior surface, e.g., silane groups in silica
capillary channels, the polymer will include monomer subunits that are
negatively charged. In accordance with the present invention, the polymer
will preferably not include any charged monomer subunits that have a
charge opposite to the charge on the interior surface of the capillary
channel. In preferred aspects, the polymer has a percent charge of between
about 0.01% and about 1% , more preferably, between about 0.01% and about
0.5%, and still more preferably between about 0.05% and 0.5%, and often
between about 0.05% and 0.2%. As noted above, in preferred aspects, the
present invention utilizes silica based substrates, e.g., planar
substrates or capillaries. As such, also in preferred aspects, the
polymers used in accordance with the invention are negatively charged, as
is the interior surface of the capillary channel.
As used herein, the "percent charge" of a polymer refers to the molar
percent of charged monomer units to total monomer subunits used in the
synthesis of the polymer. Thus, if the synthesis reaction is carried out
by mixing 1 mmol of charged subunit and 99 mmol of | | |
