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Description  |
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FIELD OF THE INVENTION
The present invention relates to capillary electrophoresis, and in
particular, to methods for achieving controlled electroosmotic flow rates
in a capillary tube, for enhancing electrophoretic separation of
biomolecules.
REFERENCES
Cohen, A. S., et al, Anal Chem, 59:1021 (1987).
Cohen, A. S., et al, J. Chromatography, 458:323 (1988).
Compton, S. W., et al BioTechniques, 6(5):432 (1988).
Herrin, B. J., J Colloid Interface Sci, 115(1):46 (1987).
Kaspar, T. J., et al, J Chromatography, 458:303 (1988).
Lauer, H. H., Anal Chem, 58:166 (1985).
McCormick, R. W., Anal Chem, 60(21):2322 (1988).
BACKGROUND OF THE INVENTION
Capillary electrophoresis (CE) has been proposed for rapid fractionation of
a variety of biomolecules (Cohen, 1987, 1988, Compton, Kaspar). In the
usual CE procedure, the capillary tube is filled with an electrophoresis
medium, a small sample volume is drawn into one end of the tube, and an
electric field is placed across the tube to draw the sample through the
medium. The electrophoretic medium may be a non-flowable polymer or gel
material such as agarose gel, but, for many types of separation, may be a
flowable fluid medium. Electrophoretic separation of proteins in a fluid
electrophoretic medium, based on the differential charge density of the
protein species, has been reported (Lauer). For fractionation of nucleic
acid species (which have similar charge densities, and therefore must be
separated on the basis of size alone) it has been found that
high-resolution fractionation can be achieved in a fluid electrophoretic
medium containing high molecular weight polymers. This method is described
in co-owned U.S. patent application for "Nucleic Acid Fractionation by
Counter-Migration Capillary Electrophoresis", Ser. No. 390,631, filed Aug.
7, 1989.
When CE is carried out using a fluid electrophoretic medium, the medium
itself may undergo bulk flow migration through the capillary tube toward
one of the electrodes. This electroosmotic flow is due to a charge
shielding effect produced at the capillary wall interface. In the case of
standard fused silica capillary tubes, which carry negatively charged
silane groups, the charge shielding produces a cylindrical "shell" of
positively charged ions in the electrophoresis medium near the surface
wall. This shell, in turn, causes the bulk flow medium to assume the
character of a positively charged column of fluid, and migrate toward the
cathodic electrode at an electroosmotic flow rate which is dependent on
the thickness (Debye length) of the shell.
Electroosmotic flow rate may provide a important variable which can be
optimized to improve separation among two or more similar species, as has
been described in the above-cited patent application. In particular, when
CE is carried out under conditions in which electroosmotic flow occurs in
one direction, and the migration of the species to be separated is in an
opposite direction, the effective column length for separation for any
given species can be made extremely long by making the electroosmotic flow
rate in one direction nearly equal to the electrophoretic migration rate
of that species in the opposite direction.
Heretofore, attempts to modulate or control electroosmotic flow rate in CE
have been limited. In one approach, the pH of the electrophoretic medium
is made sufficiently low, e.g., less than pH 2-4, to protonate charged
surface groups, and thus reduce surface charge density This approach is
not applicable to many proteins where low-pH denaturation effects can
occur.
It has also been proposed to include in the electrophoretic buffer, a
charged agent which can bind to the surface at a given equilibrium
constant, to mask surface charge, and thus reduce electroosmotic flow.
This approach is severely limited by the problem of the charged agent
binding to the species to be separated, thus altering the charge density
and migration characteristics of these species Also, the concentration of
binding compound must be calibrated by trial and error.
Attempts to reduce or eliminate electroosmotic flow by covalently
derivatizing the charged surface groups in a CE tube with neutral or
positively charged agents has also been reported. This approach suffers
from the difficulty in calibrating the reaction conditions to achieve a
desired electroosmotic flow. In addition, the derivitization reaction is
irreversible, i.e., the tube cannot be recoated to achieve other selected
electroosmotic flow rates.
SUMMARY OF THE INVENTION
It is one general object of the invention to provide a rapid, simple method
for achieving a selected electroosmotic flow rate in a CE tube.
A more specific object is to provide such a method which is easily
performed, compatible with both protein and nucleic acid CE fractionation,
and which may be carried out in a manner which results in either
reversible or irreversible surface charge densities in a CE tube.
The invention includes, in one aspect, a method of achieving selected
electroosmotic flow characteristics in a capillary tube having charged
surface groups. The tube is connected between anodic and cathodic
electrolyte reservoirs, and an electric field is placed across the
reservoirs to produce electroosmotic flow within the tube. During
electroosmotic flow, a compound capable of stably altering the surface
charge of the tube is drawn into and through the tube, and the
electroosmotic flow rate within the tube is monitored Drawing the compound
into and through the tube is continued until a desired electroosmotic flow
rate in the tube, as determined from the monitoring, is achieved.
The electroosmotic flow rate through the tube is preferably monitored by
introducing into the tube, at selected time intervals, a series of pulses
of a flow marker whose travel through the tube can be used to monitor the
electroosmotic flow rate of a band of fluid in the tube containing the
marker solution.
In one general embodiment, the capillary tube is a fused silica tube having
negatively charged surface silane groups, and the charge-altering compound
is a polymer containing regularly spaced, charged amine groups, and
preferably a hydrophobic polymer with quaternary amine charged groups,
such as the polymer polybrene.
In another general embodiment, the capillary tube is a glass tube having
positively charged amine groups, and the charge-altering compound is a
negatively charged polymer, such as a polymer of polysulfonic acid,
polycarboxylic acid, polyphosphonic acid, or polyphosphoric acid.
Where a charged, hydrophobic polymer, such as a hydrophobic polyamine, is
used, the method may be practiced to achieve a selected degree of
overcoating which produces electroosmotic flow in a reverse direction. In
this method, the polymer is first drawn through the tube in the initial
direction of electroosmotic flow, until the charge on the surface walls is
neutralized and electroosmotic flow in the initial direction ceases.
Thereafter, the compound is drawn into and through the tube in the same
direction by electrophoresis, until electroosmotic flow within the tube in
the opposite direction reaches a selected rate. This method is useful for
separating positively charged proteins since (a) the net positive charge
of the tube walls prevents nonspecific electrostatic protein binding to
the wall surfaces, and (b) the reverse-direction rate of electroosmotic
flow can be selected to optimize protein separation.
The method of the invention may further include a step for producing a
capillary tube having a selected density of covalently attached charged
groups on the surface of the capillary wall, using a compound which has
chemical groups both for masking wall surface charge and for covalent
attachment to reactive chemical groups on the tube wall. After achieving
the desired electroosmotic flow, the coated tube is treated with a
coupling agent effective to couple the compound covalently to the surface
walls.
In another aspect, the invention includes a CE tube formed by the method of
the invention. The tube is characterized by a (a) selected electroosmotic
flow, in a given electrophoresis medium, and (b) a coating of a charged
polymer agent. In one preferred embodiment, the charged agent is a
hydrophobic polyquaternary amine polymer.
These and other objects and features of the invention will become more
fully apparent when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram of a capillary electrophoresis system used in
practicing the method of the present invention;
FIG. 2 is a schematic view of a capillary electrophoresis system designed
for operation simultaneously in both a pulsed and constant-voltage mode;
pulsed and constant-voltage mode;
FIG. 3 is an enlarged, fragmentary portion of a capillary electrophoresis
tube, illustrating electroosmotic flow (e) in a left-to-right direction,
and fragment migration (m.sub.1, m.sub.2, m.sub.3) in a right-to-left
direction;
FIG. 4 illustrates the principle of flow-rate controlled surface-charge
coating (FCSC) in accordance with the invention;
FIG. 5 shows an optical scan of marker pulses introduced at a constant time
interval, showing the slowed rate of electroosmotic flow during an FCSC
procedure;
FIG. 6 is a plot of electroosmotic flow rate as a function of coating time
with 0.0005% polybrene as coating agent;
FIG. 7 is a plot of electroosmotic flow rate as a function of coating time
using polybrene at two polybrene concentrations as coating agents;
FIG. 8 shows the equilibrium saturation of electroosmotic flow rate as a
function of coating time when spermine is used as coating agent;
FIG. 9 shows the equilibrium saturation of electroosmotic flow rate as a
function of coating time when dodecyl trimethyl ammonium bromide is used
as a coating agent;
FIG. 10 is an CE electropherogram of lactate dehydrogenase in an uncoated
CE tube;
FIG. 11 is a CE electropherogram of lactate dehydrogenase in a CE tube
coated with polybrene;
FIG. 12 demonstrates the resolution of five acetylated forms of histone H4
by CE using a polybrene-coated capillary tube;
FIG. 13 illustrates the separation of two species of RNase T1;
FIG. 14 shows the electropherogram generated from a CE run of DNA in the
absence of NaCl in a capillary tube coated with polybrene;
FIG. 15 shows the electropherogram generated from a capillary
electrophoresis run of DNA in the presence of 10 mM NaCl in a capillary
tube coated with polybrene;
FIG. 16 shows the electropherogram generated from a CE run of DNA in the
presence of 20 mM NaCl in a capillary tube coated with polybrene;
FIG. 17 shows the electropherogram generated from a CE run of two species
of RNase T1 in the absence of salt in a capillary tube coated with
polybrene; and
FIG. 18 shows the resolution of the two RNase species in the presence of 30
mM NaCl in a capillary tube coated with polybrene.
DETAILED DESCRIPTION OF THE INVENTION
I. Capillary Electrophoresis System
FIG. 1 is a simplified schematic view of a capillary electrophoresis (CE)
system 20 suitable for practicing the flow-rate controlled surface-charge
coating (FCSC) method of the invention. The system is also suitable for
carrying out electrophoretic separations using tubes prepared by the FCSC
method. The system includes a capillary tube 22 having a length preferably
between about 10-200 cm, typically less than about 100 cm, and an inner
diameter of preferably between about 25-200 .mu.m (microns), typically
about 50 .mu.m. In the embodiment shown, the tube is supported in a
horizontal position and has downwardly bent end regions.
The inner surface of the tube has chemical groups which are either
negatively or positively charged, typically at a pH of between about 2-11.
The surface chemical groups may be an inherent property of the capillary
material, such as is the case for a fused silica tube which has surface
silane groups resulting in a negative charge. Alternatively, or in
addition, the capillary walls may be treated with known derivatization
reagents for attachment of chemical groups, such as quaternary amines, to
the inner capillary walls, or with known positively charged
surface-coating agents. More generally, the capillary tube may be any tube
or channel capable of supporting a column of buffer, preferably at a
column thickness of 200 .mu.m or less. For example, the tube may take the
form of a channel formed in a glass slide or the like, and having
negatively charged surface groups. One preferred capillary tube is a fused
silica tube having an inner diameter of 50 .mu.m and available from
Polymicro Technologies (Phoenix, AZ), and a negatively charged surface
wall.
An anodic reservoir 26 in the system contains an electrolytic solution 28
which is drawn through the tube by electroosmotic flow (Section II) with
the application of an electric field across the tube ends. The anodic end
of the tube, indicated at 22a, is immersed in the solution, as shown,
during electrophoresis.
A reservoir 30 in the system may contain a marker solution, for use during
the FCSC method, or may contain a sample of molecules to be separated,
during an electrophoretic separation. Preferably the marker or sample
material is dissolved in the electrolytic solution or in water. The two
anodic reservoirs may be carried on a carousel or the like, for placement
at a position in which the lower anodic end of the tube can be immersed in
the reservoir fluid. Although not shown here, the carousel may carry
additional reservoirs containing solutions for cleaning and flushing the
tube between electrophoretic runs or different solutions, where two or
more solutions are employed in a single electrophoretic fractionation
method.
The opposite, cathodic end of the tube, indicated at 22b, is sealed within
a cathodic reservoir 32 and is immersed in an cathodic electrolyte
solution 34 contained in the reservoir, as shown. A second tube 38 in the
reservoir is connected to a finely-controlled vacuum system (not shown)
for drawing fluid, (e.g., washing and cleaning solutions, marker solution,
and electrophoresis buffer solution) through the tube and for loading the
macromolecule sample material in reservoir 30 into the tube.
A high voltage supply 40 in the system is connected to the anodic and
cathodic reservoirs as shown, for applying a selected electric potential
between the two reservoirs. The power supply leads are connected to
platinum electrodes 41, 42 in the anodic and cathodic reservoirs,
respectively. The power supply may be designed for applying a constant
voltage (DC) across the electrodes, preferably at a voltage setting of
between 5-50 KV. Alternatively, or in addition, the power supply may be
designed to apply a selected-frequency, pulsed voltage between the
reservoirs. The polarity of power supply output can be reversed. In
general, the shorter the capillary tube, the higher the electric field
strength that can be applied, and the more rapid the electrophoretic
separation.
When operated in a pulsed voltage mode, the power supply preferably outputs
a square wave pulse at an adjustable frequency of about 50 Hz up to a KHz
range, and an rms voltage output of about 10-30 KV. Higher pulse
frequencies, even into the MHz range may be suitable for some
applications.
Completing the description of the system shown in FIG. 1, a detector 44 in
the system is positioned adjacent the cathodic end of the tube, for
optically monitoring marker or sample material migrating through an
optical detection zone 46 in the tube. The detector may be designed either
for UV absorption detection and/or for fluorescence emission detection. UV
absorbance is typically carried out at 200-280 nm, using, for example, a
Kratos 783 UV absorbance detector which has been modified by Applied
Biosystems (Foster City, CA.), by replacing the flow cell with a capillary
holder. Fluorescence emission detection is preferably carried out at a
selected excitation wavelength which is adjustable between about 200-500
nm, depending on the fluorescent species associated with the marker or
sample material, as discussed below One exemplary fluorescence detector is
an HP1046A detector available from HewlettPackard (Palo Alto, CA), and
modified as above for capillary tube detection. The detector is connected
to an integrator/plotter 45 for recording electrophoretic peaks.
Using the detector shown in FIG. 1, the electroosmotic flow rate measured
during the FCSC method is determined by calculating the time required for
a marker band to travel from the upstream end (end 22a) of the tube to the
detection zone tube, near the tube's downstream end. The electroosmotic
flow rate detected in this manner thus represents an average of the
instantaneous flow rates at the time the marker is introduced into the
tube and at the time the marker is detected. It will be appreciated that
the system can be modified to allow determination of an instantaneous flow
rate, by placing directly upstream of the detector, a T-tube through which
marker can be periodically introduced into the tube.
FIG. 2 shows a fragmentary view of an electrophoretic system 50 which can
be operated in both a pulsed and constant-voltage mode. The capillary tube
52 in the system has a small-clearance break 54 adjacent and upstream of
the detection zone, indicated at 56. The tube sections on either side of
the break are coupled by a porous glass sleeve 58 which allows electrolyte
migration into and out of the tube. The coupled portion of the tube is
sealed within a reservoir 60 filled with a suitable electrolyte solution
62. A grounded electrode 64 in the reservoir is connected to the
high-voltage side of a pulsed-voltage power supply 66 whose negative side
is in communication with a suitable cathodic reservoir. The grounded
electrode 64 is connected to the high-voltage side of a DC power supply 68
whose negative side is in communication with a suitable anodic reservoir.
II. Electroosmotic Flow
This section describes the phenomenon of electroosmotic flow. The
phenomenon is exploited both in the FCSC method of the invention, for
achieving a desired surface charge coating in the electrophoresis tubes,
and in an electrophoretic separation of sample material.
FIG. 3 shows an enlarged, fragmentary portion of a capillary
electrophoresis tube 70. As seen in the figure, the negatively charged
groups on the inner tube wall, indicated by "-" symbols, are shielded by
positively charged ions in the polymer solution, essentially forming a
positively charged shell about the column of fluid in the tube. Under the
influence of an electric field, this column of polymer solution in the
medium (which is surrounded by a shell of positive charges) is drawn
electroosmotically in the direction of negative or low potential. The rate
of electroosmotic flow in the tube is indicated by the arrow e in the
figure (arrow e may be thought of as a vector with a magnitude e and a
direction along the axis of the tube). The electroosmotic flow rate e in a
capillary tube can be described by the equation:
##EQU1##
where .epsilon., .eta., .zeta., and E are the permittivity of the fluid,
its viscosity, the zeta potential, and the electrical field strength,
respectively.
The zeta potential, .zeta., as it applies to a charged wall surface,
describes the potential across the interfacial double layer between the
charged wall surface and the "inner surface" of the charged shell
corresponding to the radius of shear of the shell. The .zeta. potential is
directly dependent on the net charge of the wall surface, and can be
increased or decreased by increasing or decreasing the wall surface charge
density, respectively.
FIG. 3 also illustrates how electrophoretic separation of sample species,
such as the three negatively charged species shown at F.sub.1, F.sub.2,
and F.sub.3 can be enhanced in a CE tube which is prepared, according to
the present invention, to provide a selected electroosmotic flow rate.
Here the rate of electroosmotic flow is indicate by a vector e, indicating
a magnitude e in the downstream direction in the figure. The three species
migrate electrophoretically in the opposite direction at rates by the
vectors indicated at m.sub.1, m.sub.2, and m.sub.3. The net rate of
migration of each species in the tube is the sum of the two opposing
vectors, indicated by .mu..sub.1, .mu..sub.2, and .mu..sub.3,
respectively.
It can be appreciated that the ability to separate the three species
depends on the differences between the three net migration-rate vectors
.mu..sub.1, .mu..sub.2, and .mu..sub.3. The relative differences between
these vectors, in turn, can be selectively controlled by varying e. For
example, by making e close to m.sub.3, the vector .mu..sub.3 can be made
quite small and thus allow F.sub.3 to be readily separated from the other
two species. Likewise, by making e quite close to m.sub.2, the vector
.mu..sub.2 can be made quite small and thus allow F.sub.2 to be readily
separated from F.sub.1.
The use of selected electroosmotic flow rate to optimize protein and
nucleic acid separations will be described in more detail below.
III. Flow-Rate Controlled Surface-Charge Coating (FCSC)
The principle of the FSCS method is illustrated in FIG. 4. The lines
associated with the negative (-) and positive (+) symbols represent the
electrodes. The line connecting the two reservoirs represents a capillary
tube. T represents the time required to traverse the length of the
capillary; .mu. represents the electroosmotic flow; and the arrow to the
right of .mu. represents the vector, indicating the magnitude and
direction of electroosmotic flow. At T.sub.0, for a given applied voltage,
the electroosmotic flow is .mu..sub.0. When a positively charged polymer
coating agent, for example a quaternary amine containing polymer (see
below), is introduced into the anodic reservoir, and a voltage is applied
across the tube, the net charge on the tube wall becomes progressively
masked by the coating agent, thus reducing the zeta potential and the
electroosmotic flow rate through the tube. At some time T.sub.1,
electroosmotic flow is decreased to .mu..sub.1 (denoted as a shorter
arrow). Since the electroosmotic flow rate is directly related to the
magnitude of the charge on the capillary wall, the slowing in
electroosmotic flow with increased run time is the result of the stable
coating of the capillary walls by the polymer coating agent, and the
resulting net reduction in the charge of the capillary wall. At some point
in time, T.sub.i+.DELTA.i, the surface charges of the capillary will be
neutralized, and the electroosmotic flow, .mu..sub.i+.DELTA.i, will be
zero.
FIG. 5 shows an optical scan of marker pulses obtained during a typical
FSCS method. Here the polymer coating compound used to stably mask the
negative charge of the capillary tube wall was polybrene, a polyquaternary
amine having the subunit formula
[-N.sup.+ (R.sub.2)-(CH.sub.2).sub.6 -N.sup.30 (R.sub.2)-CH.sub.2).sub.3
-].sub.2Br
where R is CH.sub.3, and the polymer has a molecular weight typically
between 5,000-10,000 daltons. Polybrene was included in the anodic
electrolyte buffer at a concentration of about 0.0005% (percent by
volume).
At periodic, even intervals--in this case, at one-minute intervals--during
electroosmotic flow, a marker pulse, typically about 5 nl, is introduced
into the anodic end of the tube, and this pulse then travels toward the
tube detection zone at a given electroosmotic flow rate. The peaks in FIG.
5 represent the marker pulses observed at the detection zone over a
several-minute period. As seen, the distance between marker pulses
increases with increasing time (toward the right in the figure),
evidencing the decreased electroosmotic flow rate. As noted above, the
flow rate at any time period is calculated as the average time of travel
of each successive marker pulse.
The average flow rate is monitored in this fashion with continued
introduction of the polymer coating agent in the tube, until a desired
electroosmotic flow rate is achieved.
FIG. 6 shows a plot of the electroosmotic flow rate V.sub.60 as a function
of coating time, as determined from an optical scan such as that seen in
FIG. 5. As seen, V.sub.60 decreases steadily, over a 60-80 minute period
with increased time of exposure of the polymer coating agent to the tube
walls, as the agent is drawn into and through the tube by electroosmotic
flow. As above, the FIG. 6 curve was obtained using an anodic electrolyte
containing 0.0005% polybrene.
When the natural log of V.sub.60 +1 is plotted against time, a linear curve
like those seen in FIG. 7 is obtained. The two curves were obtained at
0.000.5 and 0.001 volume percent polybrene, as indicated. Interestingly,
the slope obtained with the 0.001 percent concentration is just twice that
obtained at 0.0005 percent, demonstrating a well-defined concentration
effect with the polymer agent. Details of the above FCSC method employing
polybrene are given in Examples 1 and 2.
At a polybrene concentration of 0.0025 weight percent, a steep plot was
obtained, and controlling the electroosmotic flow rate as a function of
run time was difficult. It can be appreciated, therefore, that the
concentration of polymer coating agent must be such as to alter
electroosmotic flow over a relatively extended coating period.
The FCSC method described with respect to FIGS. 5-7 was carried out with
polybrene, exemplary of a hydrophobic polyquaternary amine compound.
Additional experiments carried out in support of the present invention
have shown that polymer coating agents, such as polybrene, are effective
to stably alter the charge characteristics of the tube. By this is meant
(a) the charge characteristics of the coated tube are stable in the
absence of polymer in the electrophoresis medium, and (b) the polymer is
removed only by extensive washing and/or treatment with agents, such as
surfactants or solvent agents, capable of stripping the polymer coating
agent from the wall surface.
A number of non-polymer, positively charged compounds have been examined
for their ability to produce flow-rate-controlled surface charge coating
in a fused silica tube. FIG. 8 shows the effects of FCSC with spermine, a
non-polymeric, hydrophilic primary amine, at spermine concentrations of
0.001% and 0.005 volume percent. As seen, the electroosmotic flow
(V.sub.60 ) as a function of run time plateaus rapidly, indicating that an
equilibrium between the capillary-wall-bound spermine and the spermine in
the buffer has reached equilibrium. Maintenance of the reduced V.sub.60 is
dependent on the continued presence of spermine in the run buffer and the
ionic strength of the buffer. Details of this study are given in Example
3.
A second positively charged non-polymeric compound which was examined was
dodecyl trimethyl ammonium bromide (DTAB), a non-hydrophobic quaternary
amine. The results of time-dependent coating with DTAB, and the
accompanying reduction in electroosmotic flow (V.sub.60) with increasing
run time, are shown in FIG. 9. As can be seen from the figure, DTAB,
similar to spermine, quickly reaches an equilibrium between the capillary
wall bound DTAB and the free DTAB in the buffer. Continued presence of the
DTAB was required to maintain the reduced electroosmotic flow rate.
The non-polymer compounds tested above, either hydrophilic or hydrophobic,
and either primary or quaternary amines, are thus incapable of stably
altering the charge characteristics of the coated tube.
The above results indicate that multiple-site electrostatic, such as occurs
with a charged polymer, is necessary for stably masking the surface charge
carried on the tube walls.
The charged polymer coating agent used to stably alter surface charge may
be either hydrophobic, e.g., polybrene, or relatively hydrophilic. A
hydrophobic polymer provides the potential for interacting (binding)
between polymer molecules and this binding interaction may be exploited in
two ways. First, the capillary tube can be overcoated, for use in
producing a net positive charge on the capillary wall, as will be
described in Section IV below.
Secondly, the electroosmotic flow of a tube coated (or even overcoated)
with a hydrophobic polymer can be controlled by drawing a surfactant or
solvent agent, such as ethylene glycol, through the tube by electroosmotic
flow, at a surfactant or solvent concentration which causes a gradual
removal of hydrophobic polymer from the tube wall, and a concomitant
increase in electroosmotic flow. This approach represents a second general
embodiment of the invention in which (a) the charge on the capillary tube
is due, in part, to a charged polymer coating agent, and (b) the compound
used to stably alter the charge on the capillary tube is effective to
remove the charged coating at a slow, controlled rate.
In this regard it is noted that when using a hydrophobic polymer coating
agent in the FCSC method to partially mask surface charge, it may be
necessary to include an agent, such as ethylene glycol, to minimize
polymer interaction. For example, in the FCSC procedures described above
involving a polybrene coating polymer, the coating solution included 5
volume percent ethylene glycol.
The same considerations discussed above for a capillary tube with
negatively charged wall surface groups also apply to a tube with
positively charged surface groups, for example, a tube derivatized with
amine groups. A stable coating agent suitable for use in reducing surface
wall charge is a negatively charged polymer, e.g., a polysulfonic acid,
polycarboxylic acid, polyphosphonic acid, or polyphosphoric acid polymer.
Where the negatively charged polymer is hydrophobic, overcoating and
selective polymer removal by a surfactant or solvent agent can be
incorporated into the method for achieving a desired electroosmotic flow
rate, by the FCSC method.
In another general embodiment, the coating agent may be a zwitterionic
polymer, i.e., a polymer containing both positive and negative charges
Here the relative concentrations of the two charge groups in the coating
agent can be adjusted to achieve a desired net charge, and therefore a
desired electroosmotic flow rate, at a selected coating level.
The ability to achieve a selected electroosmotic flow rate has important
applications to CE separation methods. For example, for any given
separation application, appropriate voltage and electroosmotic flow
conditions which maximize separation can be determined (see Examples 5-8)
and then routinely and reproducibly used to effect CE separation.
Further, for repetitive separation applications, such as in a clinical
setting, the capillary tube can be covalently modified to result in a
fixed electroosmotic flow. This can be accomplished in one of two ways.
First, the capillary tube can be coated with a polymer, such as polybrene,
the exposed negatively-charged silane sites can be activated using a
bifunctional reagent, then a second coating agent can be applied to the
column and covalently attached to the capillary via the bifunctional
reagent. Further, the selected flow rate can be established with the
polymer, such as polybrene, and then the polymer coating fixed to the
capillary tube by dehydration, for example, by baking or chemical
dehydration such as by exposure to methanol. Alternatively, the polybrene
can be modified to contain a group which can be covalently coupled to the
silane group of the capillary wall using a suitable coupling agent.
IV. Applications to Protein Capillary Electrophoresis
A. Overcoating to Block Interactions with the Capillary Wall
A major limitation of the application of capillary electrophoresis to the
separation of proteins is that many proteins have a net negative charge,
resulting in non-specific protein-binding to the capillary wall. As noted
above, reducing pH to protonate the negative groups is impractical, since
many proteins are denatured below a pH required for protonation of tube
wall charge groups.
The difficulty of separation of positively charged proteins by CE is
demonstrated by the separation procedure described in Example 5 and
illustrated in FIG. 10. Three isoforms of lactate dehydrogenase (LDH)
derived from rabbit muscle were loaded onto a capillary under the
conditions described in Example 5. FIG. 10, which is an electropherogram
the CE, shows that there is no recovery of the loaded LDH, indicating that
the protein was bound non-specifically to the capillary tube wall.
In order to reverse the charge of the capillary wall, the capillary was
over-coated with polybrene. Over-coating with polybrene was accomplished
by electrocoating with polybrene at a selected concentration until the
electroosmotic flow, .mu., decreased to zero. The polymer solution did not
contain an agent, such as ethylene glycol, for limiting polymer
hydrophobic binding interactions.
At this point, additional polybrene is drawn into the capillary tube by
electrophoretic movement only. As additional polybrene is drawn into and
through the tube, the tube wall becomes overcoated, due to hydrophobic
interactions between the surface-bound polymers, as discussed above. As
this occurs, the capillary wall acquires a net positive charge, producing
electroosmotic flow in the direction opposite to the flow of polybrene
molecules through the tube. The rate of opposite-direction electroosmotic
flow (toward the anode) increases as the degree of overcoating increases,
and this rate can be monitored, as above, until a desired
reverse-direction flow rate is achieved. At some point, the rate of
reverse-direction electroosmotic flow will become greater than the rate of
electrophoretic movement of the coating agent through the tube, and the
electroosmotic flow rate will stabilize.
Electrophoresis of positively charged protein is then carried out by
reversing the polarity of the CE system, so that the protein is introduced
at the cathodic side of the tube. In this configuration, the direction of
electroosmotic flow is in a cathode-to-anode direction, and the
electrophoretic migration of positively charged proteins is in the
opposite direction. Accordingly, and as described with reference to FIG.
3, the electroosmotic flow rate can be selected, with respect to the
opposite-direction electrophoretic migration rate of a selected protein,
to optimize separation of that protein from other protein species in the
sample.
FIG. 11 illustrates the results of a capillary electrophoresis run
performed under the same conditions as those used for FIG. 10, but where
the capillary was overcoated with polybrene, and the electric field
polarity was reversed. As can be seen from FIG. 11, this capillary
electrophoresis system, using a capillary tube over-coated with polybrene,
provides an extremely efficient separation of the three isoforms of LDH.
CE was also performed using, as a sample, the highly basic protein Histone
H4 in five acetylated forms. Without overcoating of the capillary with
polybrene these proteins do not traverse the capillary. By contrast, when
a polybrene over-coated capillary is used for the electrophoresis the
system is capable of resolving the five acetylated forms of the protein
(Example 5; FIG. 12). Electrophoresis was carried out at pH 6.6.
The ability to separate basic proteins in the capillary electrophoresis
system, at pH level of between 4 and 9, provides a valuable technique for
the analysis of proteins, particularly when only small quantities of the
proteins are available.
B. Capillary electrophoresis of proteins using selected electroosmotic flow
rates
For negatively charged proteins, no charge-attraction interactions exist
between the capillary wall and the protein. The separation of these
proteins is thus based on a combination of electroosmotic flow and
electrophoretic migration of the proteins in the direction opposing
electroosmotic flow. Accordingly, it is useful to be able to adjust
electroosmotic flow to optimize protein separation by carrying out the
separation at a selected V.sub.60.
One example of the use of the FCSC method to achieve separation of a
negatively charged protein is shown in FIG. 13 (Example 6). The capillary
tube in this example was pre-coated for 5 minutes using 0.001% polybrene
and 5% polyethylene glycol minimize hydrophobic interactions which may
result in localized unshielded positive charges, followed by loading 2.5
ng of an equal-part mixture of Ribonuclease T1 (RNase T1), which has a net
negative charge, and a recombinantly created mutant species having a
glutamine to lysine substitution, i.e., one additional positive charge.
The electropherogram, shown in FIG. 13, clearly demonstrates the ability
of the system to resolve these closely-related proteins. The two species
of RNase T1 are the peaks 14.26 and 15.96 in the figure.
C. Ion-exchange Capillary Electrophoresis of Negatively-Charged Proteins
Another type of separation which can be achieved using capillary tubes
coated in accordance with the present invention involves a competition
between salt and the charged protein for charge centers on the capillary
wall. This competition is illustrated for RNase T1 and a mutant species
(described above) in FIGS. 17 and 18. The capillary tube in this example
was partially coated with polybrene, under overcoating conditions (in the
absence of ethylene glycol), and the sample mixture of RNase T1 species
was loaded on the capillary using 10 mM sodium citrate buffer (pH=6.6) as
the electrophoresis buffer (Example 8). The electropherogram resulting
from this run is shown in FIG. 17. In this figure, there are no peaks
corresponding to either RNase T1 species. The RNase species are, most
likely, retained on the column by non-specific charge interactions with
the polybrene. When the capillary tube was washed, and the partially
coated tube was loaded with t | | |
