Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-05-06
2001-08-21
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S465000, C204S600000, C204S615000, C210S198200, C210S656000, C073S061530
Reexamination Certificate
active
06277258
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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.
CACE is only one member of a family of electrophoretic focusing techniques which can be described by the simple flux equation,
N
p
,
x
=
-
D
p
ⅆ
c
p
ⅆ
x
+
(
⟨
u
p
,
x
⟩
+
z
p
ω
p
I
x
σ
)
c
p
=
0
(
1
)
where N
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
p
is a diffusion or dispersion coefficient, <u
p,x
> is the apparent chromatographic protein velocity along the x-axis, z
p
is the protein charge, &ohgr;
p
is the protein mobility, I
x
is the current density and &sgr; 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.
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
p
=0, proteins will focus at the point where the net charge on the protein vanishes, i.e., z
p
=0, as is the case with isoelectric focusing (IEF); (2) in a gradient in u
p,x
with z
p
, I and &sgr; held constant, which corresponds to CACE; (3) in a gradient in &ohgr;
p
with u
p,x
z
p
, I and &sgr; constant, e.g., focusing a protein in a urea gradient, a technique which is still untested. With u
p
held constant there are still two ways left to focus proteins: by forming gradients in I or &sgr;, both of which generate gradients in the electric field.
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).
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-&mgr;m size exclusion (SEC) packing.
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.
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.
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).
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
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.
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.
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 mod
Huang Zheng
Ivory Cornelius F.
Schuetze Fred J.
Christensen O'Connor Johnson & Kindness PLLC
Starsiak Jr. John S.
Warden Jill
Washington State University Research Foundation
LandOfFree
Device and method for focusing solutes in an electric field... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Device and method for focusing solutes in an electric field..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Device and method for focusing solutes in an electric field... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2484198