Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-04-13
2001-10-23
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S451000, C204S600000, C204S601000
Reexamination Certificate
active
06306273
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the processing of samples such as in the field of separation of biomolecules such as proteins and, in particular, separations by capillary electrophoresis and the use of capillary electrophoresis to detect such biomolecules and in the field of assays in general.
In a range of technology-based business sectors, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time-consuming, and may be entirely impracticable where only a very small quantity of a key sample or component of the analysis is available, or where a component is very costly.
Accordingly, considerable resources have been directed to developing methods for high-throughput chemical synthesis, screening, and analysis. Subsequently, considerable art has emerged, in part, from such efforts. Automated laboratory workstations have contributed significantly to advances in pharmaceutical drug discovery and genomics over the past decade. See for example, U.S. Pat. Nos. 5,104,621 and 5,356,525 (Beckman Instruments). More specifically, robotics technology has played a major role in providing a practical useful means for enabling high throughput screening (AS) methods. Reference can be made to U.S. Pat. No. 4,965,049. Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project to date.
Computerized data handling and analysis systems have also emerged with the commercial availability of high-throughput instrumentation for numerous life sciences research and development applications. Commercial software, including database and data management software, has become routine in order to efficiently handle the large amount of data being generated. Bioinformatics has emerged as an important field.
With the developments outlined above in molecular and cellular biology, combined with advancements in combinatorial chemistry, have come an exponential increase in the number of targets and compounds available for screening. In addition, many new genes and their expressed proteins will be identified by the Human Genome project and will therefore greatly expand the pool of new targets for drug discovery. Subsequently, an unprecedented interest has arisen in the development of more efficient ultra-high throughput methods and instrumentation for pharmaceutical and genomics screening applications.
In recent parallel technological developments, miniaturization of chemical analysis systems, employing semiconductor processing methods, including photolithography and other wafer fabrication techniques borrowed from the microelectronics industry, has attracted increasing attention and has progressed rapidly. The so-called “lab-chip” technology enables sample preparation and analysis to be carried out on-board microfluidic-based cassettes. Moving fluids through a network of interconnecting enclosed microchannels of capillary dimensions is possible using electrokinetic transport methods.
Application of microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both sample and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation holds great promise for contributing to advancements in life sciences research and development.
Capillary-based separations are widely used for analysis of a variety of analyte species. Numerous subtechniques, all based on electrokinetic-driven separations, have been developed. Capillary electrophoresis is one of the more popular of these techniques and can be considered to encompass a number of related separation techniques such as capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. In the context used throughout this application, the phrase “capillary electrophoresis” is used to refer to any and all of the aforementioned electrokinetic separation subtechniques.
Microfluidic devices provide fluidic networks in which biochemical reactions, sample injections and separation of reaction products can be achieved. The application of high voltage to conductive fluids within these channels leads to electroosmotic and/or electrophoretic pumping, providing both mass transport and separation of components within the sample. In these microfluidic devices, fluid flow and reagent mixing is achieved using electrokinetic transport phenomena (electroosmotic and electrophoretic). Electrokinetic transport is controlled by regulating the applied potentials at the terminus of each channel of the microfluidic device. Within the channel network, cross intersections and mixing tees are used for valving and dispensing fluids with high volumetric reproducibility (0.5% RSD). The mixing tee can be used to mix proportionately two fluid streams in ratio from 0 to 100% from either stream simply by varying the relative field strengths in the two channels.
When capillary electrophoresis is carried out using a fluid electrophoretic medium, the medium itself may undergo bulk flow migration through the capillary tube toward one of the electrodes. This electroosmotic flow is due to a charge shielding effect produced at the capillary wall interface. In the case of standard fused silica capillary tubes, which carry negatively charged silane groups, the charge shielding produces a cylindrical “shell” of positively charged ions in the electrophoresis medium near the surface wall. This shell, in turn, causes the bulk flow medium to assume the character of a positively charged column of fluid and migrate toward the cathodic electrode at an electroosmotic flow rate.
In some instances a prerequisite for conducting assays on microfluidic devices is the ability to transport large proteins (positive and negatively charged), substrates, cofactors and inhibitors or test compounds. Electroosmotic pumping has to be used to transport reagents and samples. Therefore, control of the electroosmotic flow (EOF) and capillary wall chemistry is critical to the success of a microfluidic device. It is well recognized that EOF is essential to move oppositely charged molecules in a single run; for example EOF will be used to mix a lest compound (positively charged) with a negatively charged substrate of an enzyme.
Capillary surface modifications have been an area of active research since the introduction of capillary electrophoresis. This has been prompted by the fact that basic solutes and especially proteins undergo adsorption onto the surface of capillaries. The interaction of solute with the capillary wall leads to band-broadening and in some cases irreversible adsorption. There is an enormous amount of literature describing surface modification of capillary surfaces to separate proteins. The adsorption of proteins on the walls of capillaries is a common problem in the analysis of proteins by capillary electrophoresis. Buffer additives, non-covalent coating and covalent coating have been reported to decrease protein adsorption on the walls of capillaries. Covalent coatings are especially useful in protocols that require minimal concentration of organic materials in the electrokinesis buffer. Dynamic coating is more practical to use in studies, in which separation of analyte from buffer is not important such as, for example, analysis of an enzymatic reaction.
Coatings on capillaries can be classified into two groups, one in which the modifications on the surface inhibit EOF while in others the coatings are designed to retain a certain lev
Singh Sharat
Visser Irene
Wainright Ann K.
ACLARA BioSciences, Inc.
Perkins Coie LLP
Starsiak Jr. John S.
Warden Jill
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