Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-09-11
2004-01-13
Bell, Mark L. (Department: 1755)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S452000, C204S453000, C204S604000, C073S863310, C073S863320, C073S864210, C073S864220
Reexamination Certificate
active
06676819
ABSTRACT:
REFERENCES CITED
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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
The present invention relates to interfacing two-dimensional electrophoretic separations of biomolecules, such as protein, DNA, and carbohydrates involving capillary electrophoresis (CE). Specifically, the present invention relates to interfacing multiple capillaries either fabricated on a microchip or bundled together with multiple individual capillaries as in capillary array electrophoresis (CAE) with a different channel (capillary and/or strip gel) perpendicularly to provide sample transfer to multiple capillaries simultaneously.
Usually, electrophoresis separates protein mixtures based either on their charges or on their sizes (molecular weights). By combining these two mechanisms, which are orthogonal to each other, a particularly powerful tool called two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is formed (Kenrick, K. G. and Margolis, J. Isoelectric focusing and gradient gel electrophoresis: a two-dimensional technique,
Anal. Biochem
. 1970, 204-207; O'Farrell, P. H. High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 1975, 250, 4007-4021). The modern two-dimensional electrophoresis has improved significantly with many modifications to the original technique developed almost thirty years ago. However, the general procedures remain the same, typically involving sequential separations by first dimension of isoelectric focusing (IEF) and the second dimension of slab gel electrophoresis (SGE) using sodium dodecyl sulphate (SDS).
This 2-D PAGE technology is the only technique known so far capable of separating thousands of proteins simultaneously and providing highly purified proteins. Actually, 2-D PAGE is the core technology that forms the basis for the rapid expanding field of proteomics and genomics (Wilkins, M. R., Williams, K. L., Appel, R. D. and Hochstrasser, D. F., Eds, Proteome Research: New frontiers in Functional Genomics, Springer, Berlin, 1997). Currently, proteomics and genomics heavily rely on 2-D PAGE and related technologies to separate, identify and quantitate proteins. Unfortunately, the current 2-D PAGE technique requires separate steps and is very hard to automate. Therefore, it is critical for the future development of proteomics to automate the protein separation and quantitation process. To date, very few successful results have been reported for the automation of 2-D PAGE.
Recently, capillary electrophoresis (CE) has emerged as a powerful separations technique, with applicability toward a wide range of molecules from simple atomic ions to large DNA fragments. In particular, two of the operational modes, i.e. capillary IEF (cIEF) and capillary gel electrophoresis (CGE), have become attractive alternatives to slab gel electrophoresis for biomolecule analysis, including protein separation and DNA sequencing. This is generally attributed to the fact that the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, cIEF and CGE produce faster separation with better resolution than slab gels. Especially, the sub-nanoliter sample volume requirement make these technique extremely attractive for biomedical analysis where samples are often too hard to get enough for other techniques to work. Because of the sub-nanoliter size of the samples involved, however, a challenging problem in applying this technology is to handling the samples including transferring the samples from one dimension to another.
ISOELECTRIC FOCUSING
Isoelectric focusing separation of proteins in an immobilized pH gradient (IPG) is extensively described in the art. The concept of the immobilized pH gradient (IPG) is disclosed in U.S. Pat. No. 4,130,470 and is further described in numerous publications (Bjellqvist, B., Ek, K., Postel, W., Isoelectric focusing in immobilized pH gradients: principle, methodology, and some applications,
J Biochem. Biophys. Methods
1982, 6,317-339; Gorg, A. Postel, W., Gunther, S., Weser, J., Improved horizontal two-dimensional electrophoreis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying-on transfer to the second dimension,
Electrophoresis
, 1985, 6,599-604).
It is current practice to create IPG gels in a thin planar configuration bonded to an inert plastic sheet that has been treated for chemical binding to an acrylamide gel. The IPG gel is typically formed as a rectangular plate of 0.5 mm thick, 10 to 30 cm long (in the direction of separation) and about 10 cm wide. Multiple samples can be applied to such a gel in parallel lanes, with the attendant problem of diffusion of proteins between lanes producing cross contamination. In the case where it is important that all applied protein in a given lane is recovered in that lane (as is typically the case in 2-D electrophoresis), it has proven necessary to split the gel into narrow strips, (Immobiline DryStrips, typically 3 mm wide), each of which can then be run as a separate gel. Since the protein of a sample is then confined to the volume of the gel represented by the single strip, it will all be recovered in that strip.
IEF can also be performed in capillaries (Hjerten, S., Zhu, M. D., Adaptation of the equipment for high-performance electrophoresis to isoelectric focusing,
J. Chromatogr
. 1985, 346, 265-70; Hjerten, S., Liao, J. L, Rapid separation of proteins by isoelectric focusing in the high-performance electrophoresis apparatus,
Protides Biol. Fluid
. 1986, 34, 727-30; Thormann, W., Tsai, A., Michaud, J. P., Mosher, R. A., Bier, M.,
J. Chromatogr
. 1987, 389, 75-86). In cIEF, the pH gradient is usually provided by supplying the full capillary with a mixture of the heterogeneous ampholytes and the homogeneous separation medium along with protein samples. The current cIEF suffers from two major limitations. One is the detection of the separated proteins bands. Since the whole content is restricted within the capillary, a solution with either high salt concentration or extreme pH has to be used to elute the analytes out from one of the capillary end for detection. Recently, some work to image the whole capillary for the separated bands have been reported (Fang, X., Tragas, C., Wu, J., Mao, Q., Pawliszyn, J., Recent development in capillary electric focusing with whole column imaging detection,
Electrophoresis
, 1998, 19, 2290-2295). The second limitation is the difficult to create the equivalent of IPG strips in the capillary due to geometric restriction (Hochstrasser, D., Augsburger, V., Funk, M., Appel, R., Pellegrini, C., Muller, A. F., Immobilized pH gradients in capillary tubes and two-dimensional gel electrophoresis,
Electrophoresis
, 1986, 7, 505-11). Therefore, the whole buffer system migrates, due to electroosmotic flow, during the IEF process making the focusing process difficult to reproduce. Coating the capillary surfaces with a hydrophilic layer reduces the electroosmosis and thus the buffer migration process (Bao, J., Separation of proteins by capillary electrophoresis using an epoxy based hydrophilic coating, J. Liq. Chrom. & Rel. Technol., 2000, 23, 61-78). However, most of the coatings cannot resist the extreme pHs involved in the IEF process for long. Fortunately, most of the cIEF can be accomplished within a shorter period of time due to a much higher voltage in cIEF as compared with traditional IEF process. Therefore, cIEF is still a very practical technology for protein separations.
Further, cIEF can also be performed on microchips. The details of this art have been described in details in refe
Bao James Jianmin
Liu Yaoqing Diana
Bell Mark L.
Brown Jennine
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