U.S. patent application number 09/885439 was filed with the patent office on 2002-04-18 for device and method for focusing solutes in an electric field gradient.
This patent application is currently assigned to Washington State University Research Foundation. Invention is credited to Huang, Zheng, Ivory, Cornelius F., Schuetze, Fred J..
Application Number | 20020043462 09/885439 |
Document ID | / |
Family ID | 26771056 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020043462 |
Kind Code |
A1 |
Ivory, Cornelius F. ; et
al. |
April 18, 2002 |
Device and method for focusing solutes in an electric field
gradient
Abstract
An electrophoretic device and method for focusing a charged
solute is disclosed. The device includes a first chamber for
receiving a fluid medium, the first chamber having an inlet for
introducing a first liquid to the chamber and an outlet for exiting
the first liquid from the chamber; a second chamber comprising an
electrode array, the second chamber having an inlet for introducing
a second liquid to the chamber and an outlet for exiting the second
liquid from the chamber; and a porous material separating the first
and second chambers. The device's electrode array includes a
plurality of electrodes and generates an electric field gradient
profile which can be dynamically controlled. In the method, a
charged solute is introduced into a fluid medium followed by the
application of a hydrodynamic force. Opposing the hydrodynamic
force with an electric field gradient results in solute focusing in
the fluid medium. The electric field gradient is generated by an
electrode array by individually adjusting the electrode
voltages.
Inventors: |
Ivory, Cornelius F.;
(Pullman, WA) ; Huang, Zheng; (Nantong City,
CN) ; Schuetze, Fred J.; (Pullman, WA) |
Correspondence
Address: |
CHRISTENSEN, O'CONNOR, JOHNSON, KINDNESS, PLLC
1420 FIFTH AVENUE
SUITE 2800
SEATTLE
WA
98101-2347
US
|
Assignee: |
Washington State University
Research Foundation
|
Family ID: |
26771056 |
Appl. No.: |
09/885439 |
Filed: |
June 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09885439 |
Jun 19, 2001 |
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09306645 |
May 6, 1999 |
|
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6277258 |
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60084505 |
May 6, 1998 |
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Current U.S.
Class: |
204/450 ;
204/465; 204/600; 204/615; 210/198.2; 210/656 |
Current CPC
Class: |
G01N 27/44795 20130101;
G01N 27/44743 20130101; G01N 27/44773 20130101 |
Class at
Publication: |
204/450 ;
204/465; 204/600; 204/615; 210/198.2; 210/656 |
International
Class: |
G01N 027/26; G01N
027/447 |
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A device for focusing a charged solute comprising: a first
chamber for receiving a fluid medium, the first chamber having an
inlet for introducing a first liquid to the chamber and an outlet
for exiting the first liquid from the chamber; a second chamber
comprising an electrode array, the second chamber having an inlet
for introducing a second liquid to the chamber and an outlet for
exiting the second liquid from the chamber; and a porous material
separating the first and second chambers.
2. The device of claim 1 wherein the first and second chambers are
in liquid communication when the chambers are filled with
liquid.
3. The device of claim 1 wherein the first chamber is in electrical
communication with the electrode array when the chambers are filled
with a conductive liquid.
4. The device of claim 1 wherein the electrode array comprises a
plurality of electrodes arranged linearly along the chamber
length.
5. The device of claim 4 wherein each electrode is individually
controlled.
6. The device of claim 4 wherein the electrodes are pin-shaped.
7. The device of claim 4 wherein the electrodes are
staple-shaped.
8. The device of claim 1 wherein the electrode array generates an
electric field gradient profile.
9. The device of claim 8 wherein the electric field gradient
profile can be dynamically controlled.
10. The device of claim 1 wherein the electrode array comprises an
electrode array positioned on a surface of the second chamber
opposing the porous material.
11. The device of claim 1 wherein the electrode array comprises a
first electrode array and a second electrode array, the first and
second arrays positioned on opposing surfaces of the second chamber
adjacent the porous material.
12. The device of claim 1 wherein the fluid medium comprises a
chromatography support material.
13. The device of claim 1 wherein fluid medium comprises a fluid
selected from the group consisting of a simple fluid, a complex
fluid, and a polymer solution.
14. The device of claim 1 wherein the charged solute comprises a
biological solute selected from the group consisting of a protein,
peptide, oligonucleotide, polynucleotide, and mixtures thereof.
15. The device of claim 1 wherein the charged solute comprises an
uncharged material sorbed into a charged carrier.
16. The device of claim 1 wherein the second chamber further
comprises an electrode pair, wherein the electrodes of the pair are
positioned adjacent opposing ends of the electrode array.
17. The device of claim 1 further comprising a first conduit for
introducing fluid media into the first chamber and a second conduit
for exiting fluid media from the first chamber.
18. A device for focusing a charged solute comprising: a first
block having a first trough machined therein for receiving a fluid
medium, the first trough having an inlet for introducing a first
liquid to the trough and an outlet for exiting the first liquid
from the trough; a second block having a second trough machined
therein, wherein the second block comprises a electrode array
positioned. in the trough, the second trough having an inlet for
introducing a second liquid to the trough and an outlet for exiting
the second liquid from the trough, wherein the first trough and the
second trough are substantially coincident and form a channel when
the first block is sealed to the second block; and a porous
material intermediate the first and second blocks, wherein the
porous material divides the channel formed when the first block is
sealed to the second block into a first chamber and a second
chamber, the second chamber including the electrode array.
19. The device of claim 18 wherein the first and second chambers
are in liquid communication when the chambers are filled with
liquid.
20. The device of claim 18 wherein the first chamber is in
electrical communication with the electrode array when the chambers
are filled with a conductive liquid.
21. The device of claim 18 wherein the electrode array comprises a
plurality of electrodes arranged linearly along the chamber
length.
22. The device of claim 21 wherein each electrode is individually
controlled.
23. The device of claim 21 wherein the electrodes are
pin-shaped.
24. The device of claim 21 wherein the electrodes are
staple-shaped.
25. The device of claim 18 wherein the electrode array generates an
electric field gradient profile.
26. The device of claim 25 wherein the electric field gradient
profile can be dynamically controlled.
27. The device of claim 18 wherein the electrode array comprises an
electrode array positioned on a surface of the second chamber
opposing the porous material.
28. The device of claim 18 wherein the electrode array comprises a
first electrode array and a second electrode array, the first and
second arrays positioned on opposing surfaces of the second chamber
adjacent the porous material.
29. The device of claim 18 wherein the fluid medium comprises a
chromatography support material
30. The device of claim 18 wherein fluid medium comprises a fluid
selected from the group consisting of a simple fluid, a complex
fluid, and a polymer solution.
31. The device of claim 18 wherein the charged solute comprises a
biological solute selected from the group consisting of a protein,
peptide, oligonucleotide, polynucleotide, and mixtures thereof.
32. The device of claim 18 wherein the second chamber further
comprises an electrode pair, wherein the electrodes of the pair are
positioned adjacent opposing ends of the electrode array.
33. The device of claim 18 further comprising a first conduit for
introducing fluid media into the first chamber and a second conduit
for exiting fluid media from the first chamber.
34. The device of claim 18 wherein the first block is sealed to the
second block through bolts passing through the blocks.
35. The device of claim 18 further comprising a resilient sheet
intermediate the second block and the porous material, wherein the
sheet has an aperture coincident with the first and second troughs
when the sheet is positioned intermediate the blocks
36. The device of claim 18 further comprising a sealant
intermediate the second block and the resilient sheet.
37. A method for focusing a charged solute in a fluid medium
comprising: introducing a charged solute into a fluid medium; and
applying an electric field gradient to the charged solute in the
fluid medium to cause the charged solute to focus in a region of
the medium, wherein the electric field gradient is generated by an
electrode array.
38. The method for claim 37 wherein the electric field gradient is
dynamically controlled.
39. The method of claim 37 wherein the electric field gradient is
changed during the course of focusing the charged solute.
40. The method of claim 37 wherein the fluid medium comprises a
chromatography support material.
41. The method of claim 37 wherein the fluid medium comprises a
fluid selected from the group consisting of a simple fluid, a
complex fluid, and a polymer solution.
42. The method of claim 37 wherein the charged solute comprises a
biological solute selected from the group consisting of a protein,
peptide, oligonucleotide, polynucleotide, and mixtures thereof.
43. The method of claim 37 wherein the charged solute comprises an
uncharged material sorbed into a charged carrier.
44. The method of claim 37 wherein the charged solute is a
component of a charged solute mixture.
45. The method of claim 37 wherein the electrode array comprises a
plurality of electrodes arranged linearly along an axis parallel to
direction of migration of the charged solute in the fluid
medium.
46. The method of claim 45 wherein each electrode is individually
controlled.
47. A method for focusing a charged solute in a fluid medium
comprising: introducing a charged solute into a fluid medium,
wherein the fluid medium is contained in a device comprising a
first chamber for receiving the fluid medium, the first chamber
having an inlet for introducing a first liquid to the chamber and
an outlet for exiting the first liquid from the chamber; a second
chamber comprising an electrode array, the second chamber having an
inlet for introducing a second liquid to the chamber and an outlet
for exiting the second liquid from the chamber; and a porous
material separating the first and second chambers; and applying an
electric field gradient to the charged solute in the fluid medium
to cause the charged solute to focus in a region of the medium.
48. The method of claim 47 wherein the first liquid is an eluant
buffer.
49. The method of claim 47 wherein the second liquid is a coolant
buffer.
50. The method of claim 47 wherein the first liquid is the same as
the second liquid.
51. The method of claim 47 wherein the first liquid is different
from the second liquid.
52. A method for focusing a charged solute in a fluid medium
comprising: introducing a charged solute into a fluid medium,
wherein the fluid medium is contained in a device comprising a
first block having a first trough machined therein for receiving a
fluid medium, the first trough having an inlet for introducing a
first liquid to the trough and an outlet for exiting the first
liquid from the trough; a second block having a second trough
machined therein, wherein the second block comprises an electrode
array positioned in the trough, the second trough having an inlet
for introducing a second liquid to the trough and an outlet for
exiting the second liquid from the trough, wherein the first trough
and the second trough are substantially coincident and form a
channel when the first block is sealed to the second block; and a
porous material intermediate the first and second blocks, wherein
the porous material divides the channel formed when the first block
is sealed to the second block into a first chamber and a second
chamber, the second chamber including the electrode array; and
applying an electric field gradient to the charged solute in the
fluid medium to cause the charged solute to focus in a region of
the medium.
53. The method of claim 52 wherein the first liquid is an eluant
buffer.
54. The method of claim 52 wherein the second liquid is a coolant
buffer.
55. The method of claim 52 wherein the first liquid is the same as
the second liquid.
56. The method of claim 52 wherein the first liquid is different
from the second liquid.
57. A method for focusing a charged solute comprising: introducing
a charged solute into a fluid medium; applying a hydrodynamic force
to the solute in the fluid medium; and opposing the hydrodynamic
force with an electric field gradient to provide a solute focused
in the fluid medium, wherein the electric field gradient is
generated by an electrode array.
58. The method of claim 57 wherein the electrode array comprises a
plurality of electrodes arranged linearly along an axis parallel to
direction of migration of the charged solute in the fluid
medium.
59. The method of claim 58 wherein each electrode is individually
controlled.
60. The method for claim 57 wherein the electric field gradient is
dynamically controlled.
61. The method of claim 57 wherein the electric field gradient is
changed during the course of focusing the charged solute.
62. The method of claim 57 wherein the fluid medium comprises a
chromatography support material.
63. The method of claim 57 wherein the charged solute comprises a
biological solute selected from the group consisting of a protein,
peptide, oligonucleotide, polynucleotide, and mixtures thereof.
64. A method for separating charged solutes comprising: introducing
a mixture of charged solutes into a fluid medium; applying a
hydrodynamic force to the solutes in the fluid medium; and opposing
the hydrodynamic force with an electric field gradient to separate
the charged solutes in order of their electrophoretic mobilities,
wherein the electric field gradient is generated by an electrode
array.
65. The method of claim 64 wherein each electrode is individually
controlled.
66. The method for claim 65 wherein the electric field gradient is
dynamically controlled.
67. The method of claim 64 wherein the electric field gradient is
changed during the course of focusing the charged solute.
68. The method of claim 64 wherein the fluid medium comprises a
chromatography support material.
69. The method of claim 64 wherein the charged solute comprises a
biological solute selected from the group consisting of a protein,
peptide, oligonucleotide, polynucleotide, and mixtures thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit under 35 U.S.C.
119(e) of the priority of the filing date of copending U.S.
provisional application Serial No. 60/084,505, filed May 6, 1998,
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to an electrophoretic device
and method and, more particularly to an electrophoretic device and
method that establishes and maintains an electric field gradient
using an electrode array in which the electrode voltage is
individually controlled.
BACKGROUND OF THE INVENTION
[0003] Electrophoresis is a gentle, inexpensive method of
separating molecules based on their movement in an electric field.
Electrophoresis can be carried out in free solution, e.g., an open
capillary, slit or annulus, or with the aid of a support medium,
such as a gel, polymer solution, or granular packing.
Electrophoresis requires a buffered electrolyte to maintain the
required pH and provide sufficient conductivity to allow the
passage of current.
[0004] More than a decade ago, O'Farrell described a method known
as counteracting chromatographic electrophoresis (CACE) in which
proteins could be focused at the interface between two different
gel filtration media packed into the upper and lower halves of an
electrochromatography column. Science 1985, 227, 1586-1588. The
results were soon replicated by others who found that at least one
protein, ferritin, could be concentrated beyond 100 mg/mL. Sep.
Sci. Technol. 1988, 23, 875; Sep. Purif. Methods 1989, 18, 1. This
remarkable feat was tempered by the finding that his approach
worked poorly with protein mixtures and would be difficult to scale
up. Biotechnol. Prog. 1990, 6, 21. Nevertheless, O'Farrell had
found a way to focus proteins in an electric field that did not
require the use of a pH gradient.
[0005] CACE is only one member of a family of electrophoretic
focusing techniques which can be described by the simple flux
equation, 1 N p , x = - D p c p x + ( u p , x + z p p I x ) c p = 0
( 1 )
[0006] where N.sub.p,x, the molar flux of protein along the x-axis,
is set equal to zero for stationary, focused protein bands. Eq.(1)
is composed of a dispersive term, a convective term and an
electrophoretic term where c is the protein concentration, D.sub.p
is a diffusion or dispersion coefficient, <u.sub.p,x> is the
apparent chromatographic protein velocity along the x-axis, z.sub.p
is the protein charge, .omega..sub.p is the protein mobility,
I.sub.x is the current density and .sigma. is the electrical
conductivity. In order for proteins to focus it is necessary that
at least one of the terms in parentheses vary so that their sum (1)
forms a gradient in which (2) vanishes at a single point in the
chamber. Focusing occurs at the point in the chamber where the
gradient vanishes.
[0007] Setting the sum of the terms in parentheses in eq.(1) equal
to zero, it is seen that focusing may be accomplished in at least
five different ways: (1) in a pH gradient with u.sub.p=0, proteins
will focus at the point where the net charge on the protein
vanishes, i.e., z.sub.p=0, as is the case with isoelectric focusing
(IEF); (2) in a gradient in u.sub.p,x with z.sub.p, I and .sigma.
held constant, which corresponds to CACE; (3) in a gradient in
.omega..sub.p with u.sub.p,x z.sub.p, I and .sigma. constant, e.g.,
focusing a protein in a urea gradient, a technique which is still
untested. With u.sub.p held constant there are still two ways left
to focus proteins: by forming gradients in I or .sigma., both of
which generate gradients in the electric field.
[0008] Recently, Koegler and Ivory demonstrated that charged
proteins could be separated and focused using an electric field
gradient in an electrochromatography column. J. Chromatogr., A
1996, 229, 229-236. A fluted cooling jacket was used to form a
linear gradient in the electric field which drove the proteins
against a constant flow of buffer in a packed dialysis tube. This
approach was slow and cumbersome and gave mediocre results, but it
successfully illustrated an alternative focusing technique known as
electric field gradient focusing (EFGF).
[0009] Next, Greenlee and Ivory showed that proteins would focus in
the electric field gradient formed by an axial conductivity
gradient and opposed by a constant flow of buffer. Biotechnol.
Prog. 1998, 14, 300-309. Greenlee's apparatus was far simpler to
build and operate than was Koegler's. The device was also
surprisingly fast when run in free solution, reaching equilibrium
in less than 10 min., and gave unexpectedly good results when
filled with a 40.mu.m size exclusion (SEC) packing.
[0010] Focusing can also be achieved by opposing a constant
convective velocity with a gradient in the electrophoretic velocity
of the protein. This gradient can be created by varying the net
charge on the protein (as in isoelectric focusing), by varying the
cross-sectional area through which the electric current travels, as
with electric field gradient focusing, or by varying the buffer
conductivity.
[0011] Isoelectric focusing (IEF) is a gradient focusing method
which varies the charge on a protein using a pH gradient. The
convective velocity is usually set to zero while the net charge on
the protein decreases as it approaches its isoelectric point (pI).
The protein focuses at this point since its net charge, and
therefore its electrophoretic velocity, both vanish at its pI.
[0012] Conventional IEF is usually performed in a support medium
such as agarose or polyacrylamide gel. The pH gradient is formed by
using a complex set of reagents known as carrier ampholytes which
generate a stable, linear pH gradient under the influence of an
applied electric field. Proteins migrate to the region where the
ampholyte solution pH is equal to its own pI. In gels, detection of
the focused bands involves a time consuming stain/destain
procedure, and the ampholytes should be removed before the stain is
applied. Established IEF protocols and a succinct history of its
development are given by Righetti (1983).
[0013] Despite the advances in the electrophoretic methods and
devices noted above, a need exists for electrophoretic methods and
devices that can effectively separate charged solutes, such as
protein mixtures, into their component solutes. The present
invention seeks to fulfill these needs and provides further related
advantages.
SUMMARY OF THE INVENTION
[0014] In one aspect, the present invention provides an
electrophoretic device for focusing a charged solute. The device
includes a first chamber for receiving a fluid medium, the first
chamber having an inlet for introducing a first liquid to the
chamber and an outlet for exiting the first liquid from the
chamber; a second chamber comprising an electrode array, the second
chamber having an inlet for introducing a second liquid to the
chamber and an outlet for exiting the second liquid from the
chamber; and a porous material separating the first and second
chambers. In the device, the first and second chambers are in
liquid communication when the chambers are filled with liquid and
the first chamber is in electrical communication with the electrode
array when the chambers are filled with a conductive liquid. The
device's electrode array includes a plurality of electrodes
arranged along the chamber length and each electrode is
individually controlled. The electrode array generates an electric
field gradient profile which can be dynamically controlled. The
device is useful for focusing charged solutes and for separating
mixtures of charged solutes.
[0015] In another aspect of the present invention, an
electrophoretic method for focusing a charged solute is provided.
In the method, a charged solute is applied to a fluid medium and
then a hydrodynamic force is applied to the solute in the fluid
medium. Opposing the hydrodynamic force with an electric field
gradient results in solute focusing in the fluid medium. The
electric field gradient is generated by an electrode array by
individually adjusting the electrode voltages of each element of
the array.
[0016] In accordance with the invention, the electronically
generated field can take on arbitrary shapes including exponential
profiles, steps, and even locally reversed gradients, for example,
to elute proteins. The field shape can be monitored and maintained
by computer and modified "on-the-fly" on a point-by-point basis,
both spatially and temporally. During a run the operator can
optimize the local properties of the field to tease proteins apart,
sharpen an individual band, move a band to an offtake port or set
up a moving gradient to elute one or more bands from the chamber.
With online (e.g., optical or potentiometric) monitoring in place,
the operator could be replaced by a computer programmed to detect
focused peaks and automatically adjust the field shape to optimize
the separation and, when necessary, offload products.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated by reference
to the following detailed description, when taken in conjunction
with the accompanying drawings, wherein:
[0018] FIG. 1 is a schematic drawing of a representative device
formed in accordance with the present invention;
[0019] FIG. 2 is a schematic drawing of a representative device
formed in accordance with the present invention;
[0020] FIG. 3A is an exploded view of a representative device
formed in accordance with the present invention;
[0021] FIGS. 3B-3E are schematic drawings of the components
illustrated in FIG. 3A;
[0022] FIG. 4 is an elevation view of a representative device
formed in accordance with the present invention;
[0023] FIGS. 5A and 5B are front and back plan views, respectively,
of a representative device formed in accordance with the present
invention;
[0024] FIGS. 6A and 6B are a side plan view and a cross-sectional
view of a representative device formed in accordance with the
present invention;
[0025] FIGS. 7A-D are digitized images of Phycoerythrin (PE);
Phycocyanine (PC) showing two contaminants (con); carbonic
anhydrase labeled with Texas Red (CA); and a cocktail of PE, PC,
CA, and myoglobin (MYO), respectively, focused in a ccordance with
present invention;
[0026] FIGS. 8A-C are digitized images of myoglobin (Sigma
IEF-grade) in 10 mM tris-phosphate buffer focused in accordance
with the present invention at pH 8.8, 400V, and .gradient.E=6.9,
two bands about 0.5 mm thick are separated by about 0.5 mm (A);
reducing the electric field gradient to .gradient.E=5.9 increases
resolution (B); and reducing the pH in the coolant circuit to 8.3
further improves band resolution (C);
[0027] FIG. 9 is a digitalized image of a cocktail of bovine serum
albumin labeled with bromophenol blue (bBSA), PE, and ferritin (F)
focused at pH 8.7 and .gradient.E=3.7 in accordance with the
present invention;
[0028] FIG. 10 is a graph of five simulated proteins focused in a
sharp linear current gradient which goes from zero current at the
inlet, x=0, to about 6.5 mA at the column outlet, x=6.35 cm with
the two fastest peaks overlapping near x=0.8 cm;
[0029] FIG. 11 is a graph of a parabolic gradient illustrating
complete separation of the fast protein while keeping the slower
peaks apart by flattening the front of the gradient and steepening
the rear of the gradient;
[0030] FIG. 12 is a graph of a step gradient to sharpen peaks and
set their positions precisely, the two small step changes in the
electric field located at x=1.5 and 4.5 cm allow the fast proteins
to remain separated and tightly focused;
[0031] FIG. 13 is a schematic representation of two approaches for
conducting electric field gradient focusing in accordance with the
present invention;
[0032] FIG. 14 is a schematic drawing of a representative device
formed in accordance with the present invention;
[0033] FIGS. 15A and 15B are schematic diagrams of the field
strength profile (A) and potential profile (B) of a linear field
gradient (15.5 v/cm.sup.2) formed in accordance with the present
invention;
[0034] FIG. 16 is a schematic representation of the resistance
between two adjacent electrodes in accordance with the present
invention;
[0035] FIG. 17 is a schematic diagram of a representative electric
field gradient focusing gradient control model, the blocks with
dash line frame are controller units, each of the units handles the
data acquisition and the resistance control adjacent two
electrodes;
[0036] FIG. 18 is a schematic diagram of a representative electric
field gradient focusing gradient control circuits, blocks represent
electronic boards, the thick lines represent standard ribbon
cables, data channels between the two CIO-EXP32 boards and the
CIO-DAS16Jr board are programmed rather than being physically
connected, CIO-DAS16Jr and CIO-DIO24 are plugged in extension slots
of the PC;
[0037] FIG. 19 is a circuit diagram of a representative controller
unit, pin 1 and 4 were connected to electrodes and neighboring
units, the electrical potential on the electrode is reduced by
{fraction (1/100)}, then enters amplifier LF411C where the load of
signal increased, the signal is then sent to EXP32 board through
pin 12, the control signal (pin 10, 0-5 V) from the DAC board
adjusts the current going through the optical isolator MCT275;
[0038] FIG. 20 is a circuit diagram of a representative controller
unit;
[0039] FIG. 21 is a schematic illustration of a representative DAC
board circuit diagram illustrating connections;
[0040] FIG. 22 is a schematic illustration of a representative DAC
board circuit diagram illustrating components;
[0041] FIG. 23 is a digitized image of R-phycocyanin (PC) and two
contaminants (C) focused in accordance with the present
invention;
[0042] FIG. 24 is a digitized image of CA, PE, PC, CA, MYO, and
contaminants (C) focused in accordance with present invention;
and
[0043] FIG. 25 is a schematic illustration of representative
configurations for the device formed in accordance with the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0044] The present invention provides an electrophoretic device and
method in which a charged solute such as a protein can be
simultaneously separated and concentrated by applying a first
constant force (e.g., hydrodynamic force due to buffer flow) and
opposed by a gradient in a second force (e.g., electric field).
According to the invention, a constant hydrodynamic force is
opposed by a gradient in the electric field which allows charged
molecules to focus in order of their apparent electrophoretic
mobilities. The electric field gradient is established and
maintained using an array of electrodes whose voltages are
individually monitored and adjusted by a computer-controlled
circuit board. The computer-generated electric field gradient
allows charged molecules to be focused without using a pH gradient.
For proteins, because the proteins are not focused at their pIs,
precipitates do not form, so focused concentrations in excess of 50
mg/mL are not unusual. In addition, because the field shape is
dynamically controlled from the computer on a point-by-point basis,
the field profile can be adjusted during a run to improve the
resolution of components.
[0045] In one aspect, the present invention provides an
electrophoretic device that includes a focusing chamber having an
electrode array. The focusing chamber is a divided chamber that
includes a separation chamber and an electrode chamber separated by
a porous material. Charged solute separation and focusing occurs in
the separation chamber which includes a fluid medium. The electrode
chamber includes an array of electrodes for generating a focusing
electric field gradient. The separation chamber is in liquid and
electrical communication with the electrode chamber through the
porous material. The porous material retains solutes in the
separation chamber and is permeable to certain solutes such that
the electrode chamber and separation chambers are in liquid
communication. Generally, an eluant is introduced into and flows
through the separation chamber containing the charged solute. The
eluant flow is opposed to the direction of electrophoretic
migration of the solute. The electrode array in the electrode
chamber renders the separation chamber an electrochromatography
column.
[0046] The device can optionally include, in addition to the
electrode array, an electrode pair. In this embodiment, the
electrode's of the pair are positioned adjacent opposing ends of
the electrode array.
[0047] As noted above, the focusing chamber includes an electrode
array. As used herein, the term "electrode array" refers to a
plurality of electrodes arranged so as to generate an electric
field gradient in the separation chamber for focusing a charged
solute according to its electrophoretic mobility. The electric
field generated by the electrode array can be DC, AC, or otherwise
modulated in time including asymmetric (out of phase) field
modulation. The specific nature of the electrode (i.e., size and
shape) is not critical. Suitable electrodes include pin-shaped and
staple-shaped electrodes, among others. In one embodiment, the
electrode array includes a linear array of electrodes (e.g., 50
electrodes arranged linearly) along an axis parallel to the
direction of solute migration. In addition to arrays having
electrodes arranged in line with even spacings from one to the
next, suitable arrays also include arrays in which the electrodes
are not in line and which are not separated by even spacings. Other
configurations of electrodes include two-dimensional electrode
arrays and are also within the scope of the invention.
Two-dimensional arrays include arrays having rows and columns of
electrodes. The focusing chamber can include more than one
electrode array.
[0048] Each electrode of the array is individually controlled to
provide an electric field gradient that is dynamically controlled
(i.e., maintained and adjusted during the course of solute focusing
and/or separation). Control can be manual from the device
controller, manually from the device's associated computer, or
automatically from the computer once the computer has received
feedback from a monitor (e.g., optical monitor such as a video
signal) following solute focusing. The controller can sense the
electrode's voltage and reset its voltage to its initial
setting.
[0049] The separation and electrode chambers are separated by a
porous material such that the chambers are in liquid and electrical
communication. Liquid communication refers to the ability of liquid
to pass through the porous material while (1) desired solutes are
retained in the separation chamber; (2) undesired contaminants can
be dialyzed out of the separation chamber; and desired molecules
can be dialyzed into the separation chamber. The porous material
include materials that permit the liquid communication described
above. Suitable porous materials include porous membranes such as
dialysis membranes and ion exchange membranes.
[0050] The separation chamber includes a fluid medium. As used
herein the term "fluid medium" refers to any fluid medium in which
a charged solute can be focused. Suitable fluid media include a
simple fluid (e.g., buffered water), complex fluid (e.g., a water,
acetonitrile, methanol mixture), or polymer solution (e.g., linear
polyacrylamide, polyvinyl alcohol, methyl cellulose solutions). The
fluid medium can also include a chromatography support medium or
packing. Suitable packings can be of any size or type provided that
the solute being focused does not irreversibly bind to the packing.
Packings can be porous or nonporous, pellicular or tentacle, glass,
plastic, ceramic, any nonconductor or semiconductor. Other suitable
packings include ion-exchange, affinity, reverse phase size
exclusion, gel filtration, and hydrophobic interaction
supports.
[0051] In operation, the device includes the flow of a first liquid
through the separation chamber and the flow of a second liquid
through the electrode chamber. Generally, the first liquid is an
electrophoretic eluant (e.g., buffer solution) and the second
liquid is a coolant. The first liquid can be the same as or
different from the second liquid. During focusing and depending on
the requirements of the particular separation, the composition of
either the first and/or the second liquid can be changed to achieve
the desired result. As noted above, liquid flow through the
separation chamber opposes the direction of electrophoretic
migration of the solute and can be driven by any one of a variety
of forces including electric field, pressure, vacuum, or other
motive force. In a preferred embodiment, the direction of liquid
flow through the separation chamber is opposite that through the
electrode chamber.
[0052] The device is useful in focusing and separating charged
solutes. Charged solutes that can be focused include charged
biological solutes such as proteins, peptides, oligonucleotides,
polynucleotides, and mixtures of these can be advantageously
focused in the device. Uncharged materials sorbed into charged
carriers such as micelles and liposomes can also be focused with
the device.
[0053] The device is useful in focusing, concentrating, and
separating charged solutes. The focused solutes can be eluted from
the device through one or more separation ports. Basically, the
desired solute can be focused to a region of the chamber from which
the solute can be eluted through a port. Solutes can be eluted from
the separation chamber by electric field, pressure, vacuum, or
other motive force.
[0054] The device can further include a monitoring feature which
detects solute migration. Suitable solute detection includes
optical and potentiometric methods. Integration of detection signal
with software allows automation and computer optimization of solute
loading, separation, and elution steps.
[0055] The device can be operated in a continuous mode in which
solute for focusing and/or separation is continuously loaded into
the separation chamber and focused to offlake ports where the
solutes are continuously eluted. The continuous mode is in contrast
to the batch mode in which the solute is loaded in its entirety and
then focused. The device can be operated in either mode.
[0056] The device offers scale independent control. The device is
useful in focusing solutes ranging in amount from micrograms to
grams. As described below, the basic device, controller, software,
monitoring, and circuitry permit the focusing of a wide range of
amounts of solutes.
[0057] A representative focusing chamber formed in accordance with
the present invention is shown schematically in FIG. 1. Referring
to FIG. 1, focusing chamber 10 includes separation chamber 12 and
electrode chamber 14 separated by porous member 16. Separation
chamber 12 includes elution buffer inlet 18 and outlet 20. In
operation, in one embodiment, elution buffer flows downward from
inlet 18 through chamber 12 exiting outlet 20, and coolant buffer
flows through electrode chamber 14, preferably upwardly. Electrode
chamber 14 includes an array of electrodes 22. As shown in FIG. 1,
the electrode array can be positioned on the electrode chamber
surface 24 opposing separation chamber 12 and porous member 16.
Alternatively, as shown in FIG. 2, the electrode chamber includes a
pair of electrode arrays. Referring to FIG. 2, in this embodiment,
the electrode array includes an electrode array positioned on
electrode chamber surfaces 26 and 28 adjacent separation chamber 12
and porous member 16. Device 10 can further include one or more
ports 30 for eluting solutes from the separation chamber.
[0058] A representative electrophoretic device formed in accordance
with the present invention including a focusing chamber as
described above is shown in FIGS. 3-6 FIG. 3 shows an exploded view
of the device including front and rear portions. An elevation view
of the device is shown in FIG. 4, and forward and rear plan views
of the device as illustrated in FIGS. 5A and 5B, respectively. A
cross-sectional view of a portion of a representative device
illustrating the separation chamber, porous membrane, and electrode
chamber is shown in FIG. 6.
[0059] A representative device including a focusing chamber is
shown in FIG. 3. The embodiment illustrated in FIG. 3 includes
side-by-side electrode arrays as shown in FIG. 2. Referring to FIG.
3, device 100 has basic components including first block 110 and
second block 120 separated by intermediate sheets 130 and 140.
Porous member 16 is intermediate block 110 and sheet 140. Blocks
110 and 120 and intermediate sheets 130 and 140 are formed from
machinable materials. Preferably, blocks 110 and 120 and
intermediate sheet 130 are formed from PLEXIGLAS and sheet 140 is
formed from TEFLON. In one embodiment, each component includes a
plurality of apertures 212 that are coincident with the apertures
of the other components when the components are assembled.
Apertures 212 receive bolts 214 (see FIG. 4) for securing the
assembled components and assist in sealing the assembly. As shown
in FIG. 4, the components are secured through tightening nuts 216
on bolts 214.
[0060] To form the focusing chamber, first block 110 and second
block 120 include troughs 112 and 122, respectively. Trough 122
includes the electrode arrays, each array comprising a plurality of
electrodes 22. Sheets 130 and 140 include apertures 132 and 142,
respectively. When the components are assembled, troughs 112 and
122 and apertures 132 and 142 are coincident and form a portion of
the focusing chamber 10. Intermediate sheet 140 and block 110 is
porous member 16 which divides chamber 10 into separation chamber
12 and electrode chamber 14.
[0061] First block 110 includes conduits 114 and 116 which
terminate in opposing ends of trough 112. Conduits 114 and 116
serve as inlet and outlet, respectively, for introducing fluid
media (e.g., chromatography support material) to and removing the
media from the separation chamber. First block 110 further includes
channels 118 which terminate in trough 112, which provide for
eluting focused solutes from the device through offtake ports 30
(see FIGS. 1 and 2). Channels 119 also terminate in trough 112 and
provide for introducing charged solute and eluant to the separation
chamber through inlet 18 and exiting eluant through outlet 20 (see
FIGS. 1 and 2).
[0062] Second block 120 includes conduits 215 and 217, which
terminate in opposing ends of trough 122. These conduits serve to
introduce and exit liquid flow (e.g., coolant) through the
electrode chamber. For embodiments of the device that include an
electrode pair in addition to the electrode array, second block 120
further includes channels 218 which terminate in trough 122.
Channels 218 receive electrodes 220 and 222, which like the
electrode array, are in electrical communication with liquid in the
electrode chamber when the device is in operation.
[0063] The assembled device is illustrated in FIGS. 4 and 5.
Referring to FIG. 4, device 100 includes blocks 110 and 120 and
sheets 130 and 140, and porous member 16. Conduits 114, 116, 215,
and 217, noted above, are illustrated along with connecting devices
124, 126, 224, and 226, respectively, which serve to connect the
focusing chamber with its respective supplies. Inlet connection
device 318 and outlet connecting device 320 are illustrated and
communicate with channels 119 and separation chamber inlet 18 and
outlet 20, respectively. Connector 224 leads to the device's
controller and provides current to the electrode array. The
representative device further includes first and second plates 170
and 180, respectively, which overlie the outward surfaces of blocks
110 and 120, respectively. Plates 170 and 180 can reinforce the
assembly. Plates 170 and 180 are preferably steel plates.
[0064] FIG. 6A and 6B are cross-sectional views of a portion of the
representative device described above. Referring to FIG. 6B, device
100 includes blocks 110 and 120 and sheets 130 and 140.
Intermediate block 110 and sheet 140 is porous member 16 which
divides the focusing chamber into separation chamber 12 and
electrode chamber 14. Sheet 140 serves as a spacer for adjusting
the depth of electrode chamber 14 and, accordingly, the thickness
of sheet 140 can be varied as desired. Sheet 140 is a resilient
sheet and also serves to seal block 110 to the remaining components
of the assembly.
[0065] Intermediate sheet 140 and sheet 130 is sealant layer 150.
Sealant layer 150 includes a sealant that effectively joins sheet
140 to sheet 130 and prevents liquid from escaping the electrode
chamber. Intermediate block 120 and sheet 130 is adhesive layer
160. Adhesive layer 160 includes an adhesive that effectively joins
sheet 130 to block 120.
[0066] A representative device of the invention including a
focusing chamber was formed from two blocks of 15.times.6.times.1.2
cm.sup.3 PLEXIGLAS and a 0.3 cm thick TEFLON spacer. The front
block, which houses the separation chamber (i.e., separation column
or electrochromatography column), has a trough
8.times.0.1.times.0.05 cm.sup.3 machined into it; the rear block,
which houses 50 controllable electrodes, has a trough
6.4.times.0.3.times.1.5 cm.sup.3, and the spacer has a
6.5.times.0.2 cm.sup.2 slot machined through it. The trough in the
front block is isolated from the spacer by dialysis membrane (i.e.,
porous membrane) and packed with chromatography media (e.g., 4.5
.mu.m NovaPak Diol from Waters). The rear trough and slot admit a
recirculating buffer that can have the same composition as the
running (i.e., elution) buffer, acts both as coolant, anolyte, or
cathalyte, and removes electrolysis products from electrode array.
Because the coolant is in contact with the separation column via a
dialysis membrane, the coolant can also be used to dialyze the
running buffer to exchange salts or other low molecular weight
solutes. The coolant inlet and outlet are shown in FIGS. 4 and
5.
[0067] Outside of the focusing chamber, the coolant buffer is
circulated through a glass heat-exchange reservoir submerged in an
ice bath. From here the coolant is introduced into the bottom of
the focusing chamber and is passed over the electrodes at .about.15
mL/s using a centrifugal pump (Cole-Parmer). A syringe pump
controls the flow of the running buffer through the packed bed at
15-150 .mu.L/h. The running buffer enters the column in the upper
flow inlet on the front face and exits from the lower flow outlet
on the front face. All lines are PEEK with flangeless fittings;
sample is loaded through a 10.mu.L loop on a six-port injection
valve (Upchurch).
[0068] The 50 chamber electrodes are made from 0.25-mm-o.d.
platinum wire (Aldrich Chemical), mounted in the rear PLEXIGLAS
block with a 0.05-in. pitch, and are connected to a SCSI ribbon
cable using SMS-series microstrips (Samtec). Each of the SCSI leads
is connected to its own printed-circuit (PC) monitor/controller
board mounted on the wire wrap motherboard. Each monitor/controller
board is segregated into three areas: high voltage, monitoring, and
control. The high-voltage area isolates the chamber electrode
voltages, which can be as high as 600 V, from the relatively
sensitive electronics used to measure and adjust the electrode
voltages. The monitor area of each PC board scales down the
electrode voltage by .about.100 x and sends this signal to a
commercial thermocouple board which digitizes the signal before
sending it to the computer. The computer scans all 50 electrodes,
compares these readings with the programmed profile, and sends a
digital signal to a set of 50 DACs which tell the optical isolators
to adjust the effective resistance of high-voltage line to reduce
the departure of the measured electrode voltages from the
programmed voltage profile. A complete scan/control cycle of the 50
controllers is taken every second. Each of the 50 controllers is
mounted vertically on a wire-wrapped motherboard; power to the
controllers' motherboard is drawn from the computer. A 600-V power
supply (Xantrex) provides current to the column's 50 high-voltage
electrodes via the 50 voltage controllers.
[0069] The device is operated as follows. After the recirculating
coolant has reached operating temperature and the packed column has
been cleaned, e.g., with 7 M urea, and equilibrated with running
buffer, 10 .mu.L of protein solution is injected into the column,
which has a packed volume of 28 .mu.L exposed to the 50 controlled
electrodes, using a standard sample loop. Before protein reaches
the outlet, the controller is booted using a default voltage
pattern and the power supply is brought up to a voltage in the
range 200-600 V. The operator then selects the initial electric
field gradient, and the computer program adjusts the electrode
voltages until this gradient is attained, typically less than 5
min. from a "cold" start.
[0070] The following materials were used in demonstrating the
device and method of the invention. Chemicals and biochemicals were
purchased from Sigma. Bare silica BPLC sorbents were purchased from
Sigma and Methacrylate SEC packings from Tosohaus. Various sizes of
Symmetry packings (3.5-12 .mu.m) were donated from Waters
Corporation. Fluorescent tags were purchased from Molecular Probes
(Eugene, Oreg.). Sheets of 6k MWCO dialysis membranes were
purchased from Cole-Parmer.
[0071] Typical focusing results achieved with the device of the
invention are presented in FIGS. 7-9, which are digitized images of
naturally colored or artificially labeled proteins dynamically
focused in an electric field gradient. In each of these figures,
the flow in the packed section of the column is from top to bottom
and the voltage gradient is greatest near the outlet, vanishes at
the inlet, and is linear over the 2.5-in. length of the electrode
section. In all of these experiments, the first 49 array electrodes
are anodes while the last electrode is a cathode set to ground and
the electric field strength is linear.
[0072] The proteins and run conditions used in these experiments
are listed in Table 1. Individual protein bands (FIGS. 7A-C) take
10 to 30 min. to focus depending on the flow rate of the running
buffer. The bands formed have roughly the baseline width predicted
by the linear theory discussed below and reach concentrations in
the range of 5-50 mg/mL even without subtracting the nonaccessible
volume of the packing. For example, in FIG. 7A, the phycoerythrin
band is less than 0.2 mm thick, 1.0 mm wide, and 0.5 mm deep and
contains 2.5 .mu.g of protein which translates to an apparent
focused concentration of roughly 25 mg/mL.
1TABLE 1 Run Conditions for Proteins in FIGS. 7-9.sup.a Load
Applied Protein Protein Catalog .gradient.E Flow Voltage Mass Conc
FIG. Protein (Sigma) No. pH (V/cm.sup.2) (.mu.L/h) (V) Loaded
(.mu.g) (mg/mL) 7a (R)-phycoerythrin (PE) P 0159 7.0 13.0 44 300
2.5 0.25 7b (R)-phycocyanin (PC) P 1536 7.0 13.0 42 300 5.0 0.50 7c
carbonic anhydrase (CA) C 6653 7.0 9.3 40 300 5.0 0.50 7d carbonic
anhydrase C 6653 8.0 13.0 39 300 4.4 0.44 (R)-phycoerythrin P 0159
2.8 0.28 (R)-phycocyanin P 1536 2.5 0.25 myoglobin (MYO) M 9267 5.0
0.50 8a myoglobin M 9267 8.8 6.9 100 400 10.0 1.0 8b 8.8 5.9 2.0 8c
8.4 5.9 3.0 bovine serum albumin (bBSA) 8.7 3.7 138 300 2.0 0.20
(R)-phycoerythrin P 0159 2.0 0.20 ferritin (F) F 4503 3.0 0.30
.sup.aConditions: 10 mM tris-phosphate buffer on 4.5-.mu.m
NovaPak-Diol packing.
[0073] When multiple proteins are run, as is the case in FIG. 7D,
it is sometimes difficult to set a linear field gradient where all
of the proteins can be retained in the column and baseline
separated at the same time. This is due in part to the wide
variation in mobilities in this particular group of proteins and,
to a greater extent, to the tendency of the concentrated protein
bands to merge into isotachophoretic bands if they come too close
to one another.
[0074] FIG. 8 shows how separation conditions can be modified by
the operator during a run to improve resolution. In FIG. 8A,
IEF-grade marker myoglobin is separated into two bands. In FIG. 8B,
the electric field gradient has been reduced, and a few minutes
later, the bands have moved further apart. In FIG. 8C, the pH of
the recirculating buffer/coolant has been lowered from 8.8 to 8.4
over a period of 30 min. and the distance between the bands has
increased further.
[0075] FIG. 9 is a protein cocktail containing bovine serum albumin
labeled with bromophenol blue (bBSA), PE, and ferritin and
illustrates that other groups of proteins whose mobilities are
similar can be baseline-separated with relative ease.
[0076] The results demonstrate that, in accordance with the present
invention, it is possible to establish and manipulate an electric
field gradient by using a computer-controlled array of electrodes.
In combination with a continuous counterflow of buffer, this
gradient can be used to simultaneously separate proteins whose
apparent mobilities differ by less than 10% and to focus them to
concentrations in excess of 50 mg/ml in an electrochromatography
format.
[0077] Most, if not all, members of the family of electrophoretic
focusing techniques can be described by the simple flux equation, 2
N p , x = - D p c p x + ( u p , x + z p p I x ) c p = 0 ( 1 )
[0078] where N.sub.p,x is the molar flux of protein along the
x-axis of the electric field. For focused protein bands, the flux
is set equal to zero to indicate that the bands are stationary.
Equation 1 is composed of a dispersive term, a convective term, and
an electrophoretic term where c.sub.p is the protein concentration,
D.sub.p is a diffusion or dispersion coefficient, u.sub.p,x is the
apparent hydrodynamic velocity along the x-axis, z.sub.p is the
protein charge, .omega..sub.p is the protein mobility, I.sub.x is
the current density, and .sigma. is the electrical conductivity.
For proteins to focus, it is necessary that at least one of the
terms in parentheses varies so that their sum forms a gradient
which vanishes at a discrete point in the chamber and which pushes
the protein toward that point regardless of its initial location.
Focusing occurs at the point in the chamber where the sum of the
terms in parentheses vanishes.
[0079] Setting the sum of the terms in parentheses in Equation 1
equal to zero, it is seen that focusing may be accomplished in at
least five different ways: (a) in a pH gradient with u.sub.p=0,
proteins focus at the point where the net charge on the protein
vanishes, i.e., z.sub.p=0, as is the case with isoelectric focusing
(IEF); (b) in a gradient in u.sub.p,x with z.sub.p, I, and .sigma.
held constant, which corresponds to O'Farrell's counteracting
chromatographic electrophoresis; (c) in a gradient in .omega..sub.p
with u.sub.p,x, z.sub.p, I, and .sigma. constant, e.g., focusing a
protein in a urea gradient. With u.sub.p held constant, proteins
can be focused by (d) forming gradients in I, as was done by
Koegler and Ivory, J. Chromatogr., A 1996, 229, 229-236, or (e)
forming gradients in .sigma., as was done by Greenlee and Ivory,
Biotechnol. Prog. 1998, 14, 300-309. Both of these approaches
generate gradients in the electric field similar in many respects
to the gradients generated by the instrument described above.
[0080] Setting I.sub.x=I.sub.0,x+xI.sub.1,x to form a linear
gradient in the current, the focal point is found at 3 x f = - ( u
p , x z p p I 1 , x + I 0 , x I 1 , x ) ( 2 )
[0081] and, integrating Equation 1, the concentration is given by 4
c p = M T W Z p p I 1 , x 2 D p exp [ - Z p p I 1 , x 2 D p ( x - x
f ) 2 ] ( 3 )
[0082] which yields a Gaussian distribution in the focused band.
The standard deviation, .chi., of the peak around the focal point
is then
.chi.={square root}{square root over
(.sigma.D.sub.p/z.sub.p.omega..sub.pI- .sub.1,x)} (4)
[0083] where M.sub.T is the total mass in the focusing chamber and
W is the perimeter of the chamber. Note that focused bands are made
thinner by low conductivities and steep current gradients.
Conversely, resolution, R 5 R = 1 2 u p , x 2 D p I 1 , x 1 z p , 1
p , 1 - 1 z p , 2 p , 2 ( 5 )
[0084] is improved by reducing the gradient, raising the
conductivity, and increasing the velocity of the running buffer.
The simple linear model presented above does a good job of
predicting protein location and baseline width when bands are
completely resolved. However, because the model ignores nonlinear
coupling between the electric field and the ions in solution, it
cannot accurately describe overlapping or contiguous bands. A more
detailed version of this model that can handle these situations is
given by Koegler and Ivory. Biotechnol. Prog. 1996, 12,
822-836.
[0085] The performance of the device and method of the invention
under various conditions can be simulated. The linear model can be
used to explore the advantages of electronically controlled
focusing, specifically, by adjusting the field parameters to
enhance resolution during a run. For example, a sharp linear
gradient is shown in FIG. 10 for five recombinant protein isoforms
with the electrophoretic mobilities given in Table 2 focused near
the top of the DFGF chamber. As shown in FIG. 11, these proteins
might first be moved as a unit to the center of the chamber, e.g.,
by increasing the flow rate, and then spread over the entire length
of the column by expanding the electric field so that the fastest
peak is near the chamber inlet and the slowest peak is near the
outlet By flattening and reducing the electric field gradient, the
three low-mobility peaks could be eluted from the chamber while the
two fastest peaks are retained. After switching to step changes in
the electric field the remaining two peaks, whose mobilities differ
by .about.3%, can be completely separated and individually eluted
from the chamber as shown in FIG. 12.
2TABLE 2 Simulation Electrophoretic Mobilities fast peak -1.65
.times. 10.sup.-5 cm.sup.2/V .multidot. s -1.60 .times. 10.sup.-5
cm.sup.2/V .multidot. s -1.30 .times. 10.sup.-5 cm.sup.2/V
.multidot. s -1.10 .times. 10.sup.-5 cm.sup.2/V .multidot. s slow
peak -1.00 .times. 10.sup.-5 cm.sup.2/V .multidot. s
[0086] This simulation demonstrates that it is possible to
establish and manipulate an electric field gradient by using a
computer-controlled electrode array. In combination with a
continuous flow of buffer, this gradient can be used to
simultaneously separate and focus proteins as well as other charged
molecules at concentrations in excess of 50 mg/mL in a
packed-column format.
[0087] DFGF cannot replace IEF as an analytical technique. DFGF
cannot work at the isoelectric point (pI) because the proteins'
mobilities vanish at that point. However, DFGF does effectively
extend the pH range over which focusing can take place to include
native buffers as well as non-native, denaturing, and reducing
conditions. A resultant advantage is that focusing can be
accomplished away from a protein's pI, thus avoiding the
precipitates that often form near the isoelectric point and making
it preferable to IEF as a preparative technique.
[0088] Although the above examples illustrate the use of linear
electric field gradients, the software can be modified to allow
point-by-point adjustment of the field including reversing the
field to aid in elution of fractionated bands, isolating and
mobilizing a single protein band, or stepping the gradient to
improve processing capacity. In addition, because the electronic
controller and the DFGF technique are largely independent of
chamber capacity, there is no reason DFGF cannot be applied to
other types of electrophoresis equipment operating at larger or
smaller scales.
[0089] The above examples included colored and labeled proteins. In
another embodiment, optical or other detectors can be mounted on
the chamber to provide real-time monitoring of the separation. Such
monitoring allows for computer detection of various peaks,
optimization of the separation by locally adjusting the field
gradient to tease refractory proteins apart, and then pull off
those peaks that were selected by the operator either before or
during a separation.
[0090] The principles of the method and device of the invention
will be better understood by reference to the following
discussion.
[0091] In zone electrophoresis, an electric field causes the
differential transport of charged species. Voltage is applied
across the separation path, leading to the migration of charged
species away from the starting band and along the path. Separation
develops because of differences in migration velocities, which are
proportional to the electric field, E. A simple equation is given
by
U.sub.i=.mu..sub.iE (6)
[0092] where .mu. is the electrophoretic mobility of the species
which depends on the electrical charge, which determines how
vigorously they are driven by the applied voltage, and the degree
of frictional drag, which differentially oppose their
electrophoretic motion (Mosher, R. A., Saville, D. A., and
Thormann, W., The Dynamics of Electrophoresis, VCH Publishers,
Inc., New York (1991)).
[0093] Because the charge of the species can be positive or
negative, the electrophoretic mobility has direction with respect
to the direction of the potential gradient, as does the migration
velocity, U. The surface is strongly dependent on the ionic
strength and this affects the particle mobility.
[0094] At conditions approaching infinite dilution, the
one-dimensional motion of a charged species can be described by the
flux equation. 6 N i ( x ) = D i c i x + ( u i + i E ) c i ( 7
)
[0095] where u.sub.i is the chromatographic velocity (or convective
velocity), c.sub.i is the concentration of the ion, and D.sub.i is
the diffusion coefficient of specie i. In order for species i to
focus it is necessary that at least one of the terms in parentheses
vary with respect to x so that their sum forms a gradient which
vanishes at a point in the chamber. Focusing then occurs at the
point in the chamber where these terms vanish.
[0096] In accord with this condition, there exist many ways to
accomplish focusing. First, by forming a gradient in
chromatographic velocity u.sub.i, with .mu..sub.i and E held
constant and counter-balanced with u.sub.i, which corresponds to
CACE; second, by forming a gradient in .mu..sub.i, which can be
accomplished in a pH gradient, with u.sub.i=0 and held E constant,
as in the case in IEF; third, by creating a gradient in E, with
u.sub.i and .mu..sub.i held constant, that is the case in FGF.
[0097] In all the above cases, the efficacy of separation depends
on the concentration profile of solute in the steady-state zones
and layers. It is worth reflecting on the physical origin of
steady-state conditions in separative transport. Any narrow pulse
of solute will tend to diffuse outward, and its profile can be
maintained in a steady-state condition only if some transport
process exactly balances diffusion. Such transport may be induced
by flow or external fields. The transport tends to focus solute
toward a given point, and keeps the solute compressed as a narrow
zone around that point.
[0098] For FGF, the concentration profile of solute on the simplest
field gradient can be obtained analytically. A linear electric
field gradient can be described as
E(x)=E.sub.1x+E.sub.0 (8)
[0099] where E.sub.0 is the average field strength applied on the
chamber and E.sub.1 is the increase in field strength per unit
length. If A is the cross section area of the chamber, and M.sub.1
is the total moles of the i specie. Solving Equation (6), we obtain
the concentration profile for species i 7 C 1 ( x ) = 1 2 M i i A
exp [ - ( x - i ) 2 2 i 2 ] ( 9 )
[0100] which is a Gaussian distribution with focal point .chi. and
variance .sigma..sup.2 given by 8 i = - u i + i E o i E 1 ( 10 ) i
2 = - D i i E 1 ( 11 )
[0101] The solution to this simple model indicates that, in order
to focus a protein in an electric field gradient, u.sub.i and
.mu..sub.iE.sub.1 must have opposite sign. There are two cases that
fit this condition, which are shown in FIG. 13. First, the
negatively charged proteins focus in an increasing field gradient
with the electric field in the same direction as the convective
flow of buffer (A). Second, positively charged proteins focus in a
decreasing field gradient with the electric field in opposite
direction as the convective flow (B). The amount of charge carried
on protein molecules are closely related to the pH of the buffer
and are different from species to species. The migration rate is
directly proportional to the amount of charge carried which is
different from specie to specie. Therefore, distinct stationary
accumulation zones for differently charged species are generated
along the column. In order to focus the target protein in the
chamber, the direction of electric field, the slope of field
gradient and the pH of the elution buffer must be matched.
Otherwise, the target protein will be flushed out or concentrated
at the very top of the column, allowing no separation at all.
[0102] The variance .sigma..sub.i.sup.2, which is a measure of the
width of the focused protein peak, suggests that the focused band
will be tighter and more concentrated if the diffusion coefficient
is decreased or if the slope of the field gradient is increased.
However, increasing the slope of the gradient will move the focused
bands closer together, so that resolution will decrease. There is a
trade-off between the resolution and the shape of the peak.
Equation 9 indicates that electrophoretic mobility, the convective
velocity and the field gradient determine the position of the
focused band in the column.
[0103] The present invention relates to dynamic field gradient
focusing (DFGF). Unlike the fixed field gradient design in the
prototype apparatus in Koegler's previous work, in the present
invention (see FIG. 14), dynamic electric field gradients are
created by a computer-controlled external circuit, which
manipulates the field strength between each pair of adjacent
electrodes. With the circuits and the controlling software we
developed, varying field strength along the separation chamber is
achieved. A linear electric field gradient created by the circuits
is shown in FIG. 15.
[0104] In a typical DFGF operation, the electrophoretic force on
charged species and the driving force by which the samples move
through the column are all directly opposed to each other in
direction. The driving force can be a summary of the influences of
the convective flow of the elution buffer, the chromatographic flow
and the electroosmotic flow (if the packing material surface is
charged). The combined influence on a particular specie can be
precisely canceled out to achieve a steady-state at a unique point
in the column.
[0105] The porous membrane is conductive to heat and buffer ions
but not to bulk fluid flow. With this design, the electrodes are
isolated from the packed column (i.e., separation chamber) to avoid
disruption of the laminar flow by gas generation or denaturation of
protein by contact with the electrodes. The same buffer is used for
the packed column and the electrode chamber to ensure the ion
balance between the two sides. The recycle buffer goes upward in
the electrode chamber, effectively removing the tiny gas bubbles
generated at the electrodes and acts as coolant to remove the Joule
heat generated. Another important role of the recycle buffer is to
conduct the electric field gradient through the dialysis membrane
to the packed column. In the packed column, the elution buffer is
injected from top to bottom to prevent the beads from
fluidizing.
[0106] For practical DFGF operation, Equation 8 is too simple to
predict the behavior of protein bands in the column. The
chromatographic retarding force affects the migration of the
protein sample, however, it does not affect the position of the
focused band in the packed column. Instead, it shows its effect by
reducing dispersion. In general, chromatography with a packed
column, three main independent processes contribute to band
broadening of solute zones when the migrate through the column,
namely, the unevenness of flow through the packing (eddy
diffusion), axial molecular diffusion and solute resistance to mass
transfer between phases. In DFGF, the electrophoretic behavior of
the protein molecules and buffer ions also play important
roles.
[0107] First, natural convection produced by Joule heating disturbs
the flow profile in the packed column. A temperature gradient in
the axial direction causes an uneven distribution in the viscosity,
density, and pH of the buffer, and contributes to zone broadening
in the packed bed. One might argue that this problem can be
overcome by reducing the conductivity of the carrier buffer, but
this can only go so far before the protein concentration surpasses
its solubility limit or the device develops a conductive dielectric
instability Hunter, J. B., Progress in Mathematical Modeling of
Cace, in Marcel Dekker, Inc., C. F. Ivory, Editor, 1988, Marcel
Dekker, Inc., New York. p. 875. The recycling of the coolant buffer
in the DFGF apparatus greatly improves heat dissipation. For the
thin column, we used (1 mm diameter), the resolution loss due to
Joule heating will not likely be the major problem. However, for
large scale apparatus, this should not be neglected.
[0108] The ionic strength of the buffer affects the DFGF on several
aspects: the ion concentration affects the protein interaction with
the packing materials, relatively high concentration buffer
stabilizes the protein sample and therefore avoids precipitation
and unfavorable adsorption on the surface of the packing. However,
in general, high ionic strength means high conductivity of the
buffer, which increases the heat generation and power consumption
and, for DFGF, sets a limit for the highest applicable field
strength. For charged column packing, electroosmotic flow (EOF) is
generated under the action of the electric field, and is closely
related to the ionic strength of the buffer used. In general, the
lower the ion concentration, the higher the EOF rate.
[0109] An asymmetry in band shape is frequently seen in zone
electrophoresis as well as in DFGF, which is always present when
the mobilities of sample and buffer ions are unequal. DFGF is
mainly used for the separation of high molecular weight components,
such as proteins, peptides and probably nucleic acids. In general,
the sample has smaller diffusion coefficients and electrophoretic
mobilities than the buffer ions, and, as a result, the sample zone
will often have a sharp frontal boundary. The migrational
dispersion due to the electric field is usually much larger than
diffusional dispersion. The same phenomena have been observed in
DFGF.
[0110] At high sample concentrations, the shape of the focused band
is not expected to follow the Gaussian distribution predicted by
the model. The field gradient itself will distort at the point
where proteins focus Koegler, W. S. and Ivory, C. F., "Focusing
Proteins in an Electric Field Gradient," J. Chromatography, 1996.
229:p. 229-236, the concentration profile will deviate from the
symmetrical Gaussian distribution and the bands of components with
similar mobilities will overlap.
[0111] The field gradient affects resolution and capacity. With a
shallow field gradient, more protein can be accommodated on the
column before the bands overlap, therefore, the capacity can be
increased by using a shallow gradient. The disadvantage is that
proteins with large differences in their mobilities cannot focus
simultaneously on the column Some will be flushed out, and some
will squeeze on top of the column. To solve this problem, a step
gradient can be employed. By setting one of the steps to a field
strength corresponding to the mobility of target protein, large
amounts of protein can be held in the gradient with less distortion
in gradient. At the same time, a broad range of proteins can still
focus on the same column.
[0112] Electroosmotic flow (EOF) is generated by the charges
present at the inner surface of the column or at the surface or
interior of packing beads. In free solution, as in a capillary,
electrophoresis gives rise to a bulk flow which strongly affects
the shape and width of the solute bands. EOF increases the
resolution of CE due to its uniform velocity profile in radial
direction. In a packed column, the effects of a containing wall can
be neglected if very small beads are used or the zeta potential of
the wall is the same as the packing. By visualizing the packed
column as parallel tubes with the potential of the wall being equal
to that of the particles, EOF velocity profile is flat for the
packed column. With this consideration, EOF might increase the
resolution of DFGF. However, local EOF rates near focused protein
bands may differ from the average and may cause distortion in the
flow profile, which will degrade the separation.
[0113] When charged particles like silica gel were used as the
packing, EOF, instead of the pump, is the main driving force to
recirculate the coolant buffer. EOF works on very small beads,
without generating high pressure. EOF is related to the
zeta-potential, which depends on the charged state of the surface.
One of the shortcomings of EOF pumping is that the zeta-potential
can be easily influenced by factors such as temperature, alteration
of the surface resulting from the adsorption of ions and molecules,
and the concentration and pH of the buffer electrolytes. Ionic
surfactants, such as CTAB, can provide significant change in the
EOF rate, even reversing EOF and therefore might be used to adjust
the flow rate for DFGF.
[0114] The dynamic electric field gradient focusing provided by the
present invention relies on field gradient control, which includes
hardware and software. Representative gradient control hardware and
software are discussed below.
[0115] The control circuits are designed to manipulate the field
gradient by adjusting the effective electrical resistance between
each two adjacent electrodes (see FIG. 16). in one embodiment, each
pair of electrodes is connected to one of the 50 controller units
(FIG. 17).
[0116] The electrical resistance between two adjacent electrodes
R.sub.i is determined by the sum of the resistance of three
parallel resistors, Rc.sub.i, Rp.sub.i, and Rx.sub.i. Note that the
buffer between electrodes is considered as a resistor Rc.sub.i. 9 R
1 = Rc i Rp i Rx i Rc i Rp i + Rc i Rx i + Rp i Rx i ( 12 )
[0117] The resistors Rp.sub.i are used for protective purpose and
have 1M.OMEGA. resistance. Because R.sub.p>>Rc.sub.i,
R.sub.p>>Rx.sub.1. Equation (12) can be simplified as 10 R i
= Rc i Rx i Rc i + Rx i ( 13 )
[0118] By changing each Rx.sub.i, the circuits adjust each R.sub.i
indirectly. By Ohms Law, the potential drop between two electrodes
is determined by the resistance between them if the total current
going through is constant. The potential drop between the two
adjacent electrodes is given by 11 V i = V total R i i 50 R i ( 14
)
[0119] Since the field strength is proportional to the potential
drop with the electrodes equally spaced, we can manipulate the
field strength point by point by adjusting each Rx.sub.i,
independently 12 E i = V i d = V total d R i i 50 R i ( 15 )
[0120] where d is the distance between the two adjacent electrodes.
An electric field gradient in any shape, linear or nonlinear,
continuous or stepwise, can be produced with a limitation to the
conductivity of the buffer. Note that the resistance between two
parallel-connected resistors is always less than any one of them,
in other words, R.sub.i<Rc.sub.i must be satisfied.
[0121] There is more than one group of R.sub.i that satisfies
Equation 15, in other words, different groups of Rx.sub.i can be
used to establish the same field gradient with the total current
going through the chamber arbitrarily. There is no unique
equilibrium state. To solve the problem, a small modification to
unit No. 25 is made by disabling its control function and replacing
Rp.sub.25 with a 5k.OMEGA. resistor. The total current going
through the chamber was fixed, and given by 13 I = V 25 Rp 25 Rc 25
( Rp 25 + Rc 25 ) ( 16 )
[0122] V.sub.25 has a unique value for a specific field gradient,
and can be calculated from the total potential drop across the
chamber. Rc.sub.i is determined by the conductivity of the buffer.
Therefore, there is a unique value of Rx.sub.i that satisfies
Equation 15.
[0123] Representative DFGF gradient control circuits are shown
schematically in FIG. 18. Referring to FIG. 18, the PC
monitor/controller board and the 13 bit DAC board were built in our
laboratory. Some modifications have been made for better
performance. The two thermocouple boards CIO-EXP32, the 16-channel
ADC board CIO-DAS 16/Jr and the 24-channel Digital I/O board
CIO-DIO24 were purchased from ComputerBoards, Inc. Standard SCSI
ribbon cables are used to connect all the boards. There are 50
controller units plugged into the mother board. Each unit
corresponds to one pair of electrodes. The whole system was
grounded to protect the circuits from unexpected shock.
[0124] The gradient control is accomplished with PC-controlled
circuits (see FIG. 19), which are composed of electronic circuit
boards. A circuit diagram of the controller unit is shown in FIG.
20. A logic diagram for circuit diagram for ADC board is shown in
FIG. 21. A circuit diagram for the ADC board with components
identified is shown in FIG. 22.
[0125] The circuits scan all 50 electrodes and scale the signals
down by {fraction (1/100)}. Then the signals were sent to ADC board
where 0-10V analog signals are digitized. The computer compares
these readings with the programmed gradient, then sends its
commands in digital signals to DAC board via the Digital I/O
boards. In the DAC board, the command signals are converted to 0-5V
analog signals, then sent to the 50 units on the PC
monitor/controller board. Those units adjust the current going
through the units, or we can say change the values of resistance
Rx.sub.i. Note that the Rx.sub.i do not exist physically, and they
are the resistance to current going through the chip MCT275, an
optically isolated controller. The scan/response cycle for the
circuits is set at about 0.5 sec, and could be adjusted by the
program.
[0126] A 600V DC power supply (Xantrex) supplies power to the
chamber. The power to all the boards is supplied by the
computer.
[0127] A representative device of the invention was formed as
described below and was used to: (1) focus a single protein from a
dilute solution, and (2) fractionate a protein cocktail.
[0128] A representative separation chamber was built and assembled
as described above. A 10.times.2 cm.sup.2 dialysis membrane covered
the trough to form a tube with a half-round cross sectional area.
The tube was packed with beads and serves as the separation
chamber. On the other side of the membrane, a 3 mm thick TEFLON
spacer with a 1 mm slot and a buffer chamber which was a trough
machined on a piece of PLEXIGLAS plate, with 1 mm in width and 4 mm
in depth, was arranged. The buffer chamber was a cuboid space
through which the externally cooled electrolyte buffer flows. The
recirculating buffer acts as coolant and electrolyte and removes
electrolysis products from the electrodes.
[0129] A set of 50 platinum wires (0.25 mm OD) was sealed in a row
of holes (0.05 inch between adjacent holes) in the plate with one
side contacting the buffer and the other side connected to a 50 pin
SMS-series micro strip (Samtec) which was mounted in a 2 mm deep
slot machined on the outside of the plate. Through the strips,
those electrodes were connected with external control circuits via
a 50 pin standard SCSI ribbon cable.
[0130] A dialysis membrane (MWCO 6,000) between the packed column
and buffer chamber allow ions to move in and out freely while the
charged solutes (e.g., proteins and other macromolecules) in the
column cannot penetrate the membrane. Furthermore, the dialysis
membrane isolates the column from the electrodes to avoid
disruption of the laminar flow by gas generation or denaturation of
solute (e.g., protein) by contact with electrolysis products.
[0131] The column was packed from the top with a 125-150 .mu.L/hr
flow rate using the elution buffer. For charged packing materials,
for example, silica gel, the bed was packed with a 500V reverse
field.
[0132] Inlets for the elution buffer and coolant buffer were
machined on the two PLEXIGLAS plates with corresponding interfaces
installed. Additional ports designed for packing and unpacking were
also machined at the end of the chamber.
[0133] The coolant buffer recirculates at a flow rate of about 50
L/hr between the separation chamber and a buffer reservoir in an
ice-bath. A bubble trap was arranged in the coolant buffer route to
prevent entrained gas bubbles from entering the separation chamber.
A syringe pump was employed to push the elution buffer through the
packed column at 15-150 .mu.L/hr and to generate the convective
flow that counteracts the electric field gradient. In the following
examples, 10 mM Tris-Phosphate buffer was used for both the elution
buffer and the recycle buffer. Protein sample was loaded onto the
packed column through a 6-port sample injection valve which had a
sample volume of 10 ul. All lines were PEEK with flangeless
fittings.
[0134] Dialysis membrane was purchased from Scienceware.RTM.
Bel-Art products with a 6,000 normal MWCO and 0.073 mm thickness.
Particles of different sizes (see Table 3) and different materials
(see Table 4) were been tested as packings for the separation
column. Focusing of proteins was accomplished in all the packings
tested. However, some packings provided good separation while
others did not.
[0135] Generally, the smaller the particles, the higher the
resolution. However, too small particles make it difficult to pump
the buffer through the column. For charged particles, EOF can act
as the pump. Excessive pressures can be avoided by using EOF
pumping; however, an alternative way is required to control the
flow rate, for example, by adjusting the viscosity of the buffer
and the charge density on the surface of particles.
3TABLE 3 Sizes of Symmetry Packings (Waters Corporation) Particles
Descriptions Symmetry 3.5 .mu.m 100 A 0.85 cc/gm pore volume;
Symmetry 5 .mu.m 100 A surface area = 335 m.sup.2/g; Symmetry 7
.mu.m 100 A 100 A average pore diameter; Symmetry 12 .mu.m 100 A
(.+-.25%) Particle size distribution .+-. 18.about.20% of mean
(volume) Symmetry 5 .mu.m 300 A 0.75 cc/gm pore volume; surface
area = 110 m.sup.2/g; 275-300 A nominal pore diameter; Particle
size distribution 5.5 .mu.m .+-. 18.about.20% of mean (volume)
Nova-Pak Diol 4 .mu.m 80 A 0.25 cc/gm pore volume; surface area =
120 m.sup.2/g; 80 A nominal pore diameter; acid treated to remove
metals, but not high purity silica bonded to full coverage with
Diol, no secondary end cap; Particle size distribution 4.5 .mu.m
.+-. 18% of mean (volume)
[0136]
4TABLE 4 Materials Packing Beads Packing Producer Description
TOYOPEARL.sup.[1] TosoHAAS HW-55F 45 .mu.m Size exclusion resin,
resolution factor <1.2 Particle size: 30.about.60 .mu.m
Fractionation range (MW, globular): 1.000.about.700 .times.
10.sup.3 SEC 10 .mu.m.sup.[2] Sigma TSK G3000SW Particle size: 10
.mu.m Fractionation range (MW, globular): 10.about.100 .times.
10.sup.3 Pore size: 250 A Duke 5.about.60 .mu.m Duke Sci. Corp.
Glass microspheres Duke Scientific Corporation Particle size:
5.about.60 .mu.m Duke 5.about.38 .mu.m Duke Sci. Corp. 5.about.38
fraction.sup.[3] of above particles Particle size: 5.about.38 .mu.m
5 .mu.m silica gel.sup.[3] Sigma HPLC sorbent Particle size: 5
.mu.m Pore size: 60 A Superose .RTM. 12 Sigma Prep grade Pharmacia
LKB Particle size: 20.about.40 .mu.m Fractionation range (MW,
globular): 1000.about.300 .times. 10.sup.3 .sup.[1]TSKgel SW is a
silica-based hydrophilic bonded phase for separations based on
molecular size Nonspecific interaction with proteins is minimal.
.sup.[2]TOYOPEARL HW: TOYOPEARL is totally porous, semirigid
spherical gel designed for medium and low pressure liquid
chromatography. TOYOPEARL HW gels are synthesized from hydrophilic
vinyl polymer containing numerous hydroxyl groups and are composed
exclusively of C, H and O atoms. TOYOPEARL HW is very strong
mechanically and can be used at high flow rates. .sup.[3]5.about.38
.mu.m fraction was obtained by sieving the 5.about.60 .mu.m with a
38 .mu.m metal screen. .sup.[4]Silica gel for normal phase
adsorption-partition chromatography.
[0137] Before loading a sample, the packed column was cleaned with
0.1 M NaOH and 10% Tween-20, and equilibrated with elution buffer
for at least 30 minutes. The coolant buffer in the reservoir was
brought to the operating temperature (below 8.degree. C.). A 10
.mu.L protein sample was injected into the column and, before it
reached the outlet, the controller was booted using a default
voltage pattern and the power supply was set at 150-500 V.
[0138] A global electric field gradient was selected from the
keyboard. The computer program adjusted the electrode voltages
gradually until this gradient was attained. Typically, an electric
field gradient reaches its equilibrium state within five minutes.
The protein sample focused as bands in 30 minutes and kept changing
its shape until equilibrium was reached. The pumping rate of
elution buffer and the gradient setting were adjusted to improve
resolution. Usually, colored proteins were used as samples and the
shape and position of the bands were recorded with a camera or
camcorder.
[0139] The first group of experiments was the focusing of a single
protein sample from a dilute solution to a band of more
concentrated protein to demonstrate that DFGF is capable of
focusing proteins in an electric field gradient.
[0140] The second experiment demonstrates the purification
potential and resolving power of DFGF by fractionating a protein
cocktail into isolated bands.
[0141] All the experiments were performed in a 10 mM Tris-phosphate
buffer because the buffer system has low conductivity and still has
a considerable buffer capacity. A low conductivity buffer is
preferred for DFGF. Another advantage of low conductivity buffer is
that a field gradient can be maintained more readily than in a high
conductivity buffer system.
[0142] A sample of 0.5 mg/mL R-Phycocyanin (Sigma P-1536) was
focused at a flow rate of 40 .mu.L/hr and a linear gradient of 9.3
V/cm.sup.2. The total voltage applied was 300 V, and a total
current 3.409 mA. The picture taken 2.5 hours after power-on shows
that the sample focused as three separated bands (FIG. 23). A vivid
blue-green main band (1 mm height, 42 mm from top inlet), a faint
blue band (2 mm height, 50 mm from inlet) and a faint gray band (2
mm height, 62 mm from inlet) were observed. The last two bands are
contaminants in the sample.
[0143] To demonstrate the purification potential of DFGF, a model
protein mixture (or "cocktail") was loaded onto the column. The
four-in-one cocktail proteins (0.28 mg/ml R-Phycoerythrin (PE),
0.33 mg/ml Carbonic anhydrase conjugate (CAC), 0.25 mg/ml
R-Phycocyanin (PC), 0.5 mg/ml Myoglobin, Sigma) were focused as
separate bands in the column (see FIG. 24). Focusing was
accomplished with a linear gradient of 13.0 v/cm.sup.2 and a flow
rate of about 18 .mu.l/hr. 300 V was applied across the chamber;
the current was 3.245 mA.
[0144] In all of the experiments above, focusing was carried out in
10 mM Tris-phosphate buffer at pH 7.0 (25.degree. C.). Nova-Pak
Diol silica gel beads with 4 .mu.m nominal particle size and 80 A
nominal pore size (Waters) was used for the packed column.
[0145] The resolution of the technique can be estimated from the
minimum difference in properties that allows isolation of two
adjacent bands. By measuring the distance between the two adjacent
bands in the packed column, a sample calculation can be used to
determine the difference in electromobility of two components.
[0146] For example, in the separation of the protein cocktail (FIG.
24), the distance between the two minor bands of R-Phycocyanin is 1
mm, the difference in field strength between the positions of the
two bands is about 1.3 V/cm and the average field strength is 34.6
V/cm The relative difference in electromobility between the two
bands is estimated to be about 3.8%.
[0147] In another experiment, two 0.2 mm height myoglobin (Sigma
M-1882) bands were observed focused in a 21.7 V/cm.sup.2 linear
gradient with 0.5 mm between them. Similarly, the resolution was
estimated to be about 1.3% different in electromobility.
[0148] Compared with some available electrophoresis techniques, for
instance SDS-PAGE, which can routinely isolate a discrete spectrum
of proteins whose molecular weights differ by less than 2%, the
resolving power of DFGF is very competitive.
5TABLE 5 Electromobility of Protein Samples Superficial Protein
Flow Rate Field Gradient Bands Position Sample (.mu.l/hr)
(V/cm.sup.2) (mm) CAD.sup.[1] 40.0 9.3 28.5 PE 44.0 13.0 40.0
Ferritin 39.0 3.7 34.5 42.0 PC.sup.[2] 42.0 13.0 55.0 57.0
Myoglobin.sup.[3] 37.5 3.7 17.0 20.0 .sup.[1]Dye-labeled carbonic
anhydrase by conjugating Carbonic anhydrase (Sigma) with Texas Red
- X (Molecular Probes F1uoReporter .RTM.). .sup.[2]At this
condition only two bands of R-Phycocyanin were observed.
.sup.[3]The mobility data of myoglobin (Sigma, M-9267) was obtained
in 10 mM Tris-phosphate buffer at pH 8.0.
[0149] The average protein concentration in the focused bands can
be estimated from the height of the band. For example, the
myoglobin band in FIG. 24 is about 0.5 mm in height, the volume
occupied by the focused band can be calculated from the cross
sectional area of the column. The myoglobin in the sample was
concentrated by 37 fold, from 10 .mu.L to 0.27 .mu.L. The average
concentration in the band was about 18.5 mg/ml. Subtracting the
volume of the packing, protein concentrations as high as 50 mg/ml
can be obtained in focused bands in DFGF column.
[0150] For most proteins, the solubility is lowest in the buffer
with pH equal to its pI. DFGF is generally carried out in a buffer
with pH differing from the isoelectric point (pI) of the target
proteins. For this reason, DFGF can provide highly concentrated
protein bands in a low ionic strength buffer without
precipitation.
[0151] The device of the present invention includes a focusing
chamber. As noted above, the focusing chamber can include more than
one electrode array. For example, two electrode arrays can be
associated with a single separation chamber in a configuration in
which the separation chamber is positioned in between the two
arrays. Similarly, the focusing chamber can include, for example,
four arrays positioned about a separation chamber in a
quadrupole-type configuration. Representative devices including
one, two, and four electrode arrays are illustrated schematically
in FIGS. 25A-C. Referring to FIG. 25, representative device 10
including a single electrode array (i.e., electrode chamber 14) and
a separation chamber (i.e., chamber 12) is shown in FIG. 25A. FIGS.
25B and 25C illustrate representative devices having two and four
electrode arrays arranged about a separation chamber.
[0152] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
* * * * *