Researchers Submit Patent Application, "Method and Apparatus for Precise Seletion and Extraction of a Focused Component in Isoelectric Focusing Performed in Micro-Channels", for Approval
No assignee for this patent application has been made.
News editors obtained the following quote from the background information supplied by the inventors: "Capillary electrophoresis (CE) has been established as an important separation technique in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL-nL volumes, can be achieved with CE. This is generally not possible with more conventional separatory methods, even high-performance liquid chromatography (HPLC). There are several CE separation modes in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis and capillary isoelectric focusing (cIEF).
"CE provides high-resolution and high efficiency separation and is used in proteomic research and biopharmaceutical applications. Not only proven as a powerful analytical tool, CE is also promising for application in nano and micro-fractionation collection. For instance, on-line coupling of CE with mass spectrometry (MS) is used to elucidate protein structure, and off-line CE fractionation collection is important for further characterization of proteins in connection with sequencing, peptide digesting and mapping and reaction studies.
"Various designs of microfluidic apparatus have been used for CE fraction collection. Vial collection and membrane collection of individual components of an analyte at the exit of the separation column, with or without the help of sheath fluid, has been investigated. In point-detection capillary electrophoresis, sample fractions are collected from the outlet of the separation channel after passing the detection window. Karger et al.  further developed vial collection to a fraction collector. As CE is normally run with 25 to 75 .mu.m i.d. capillaries or micro-channels, extremely small volumes of individual fractions can be expected. Therefore, exact timing is important for precise fractionation. Cross-contamination frequently occurs for closely migrating peaks due to the extremely narrow peak width and extremely small amount of eluant.
"An important CE mode, capillary isoelectric focusing (cIEF), is used for separating amphoteric substances such as peptides and proteins in a capillary or micro-channel under an electric field. Across the separation capillary or channel, voltage is applied and a pH gradient is created by carrier ampholytes that have been pre-mixed with the analyte sample, acidic at the anodic end of the channel and alkaline at the cathodic end of the channel. Each component in the analyte mixture migrates to a position in the separation channel where the surrounding pH corresponds to its isoelectric point. Therein, as zwitterions possessing no net charge, molecules of that component cease to move in the electric field. Different amphoteric components are thereby focused into narrow, stationary zones.
"cIEF is the highest resolution CE mode for charge-based separation of amphoteric substances such as proteins and peptides. It has most often been used to separate closely related proteins having subtle differences between their structures. In the cIEF separation channel, components of the analyte mixture, which evenly distribute along the whole channel before the separation process, are separated and focused into narrow, stationary, component zones.
"Uniquely narrow zones are formed using cIEF because: 1) zone broadening due to parabolic flow is not a factor in the separation process; 2) the focusing force is reverse to diffusion and 3) the electrophoresis current during focusing is low compared to other CE modes thus minimizing the effects of component zone broadening due to Joule heating. For these reasons, the analyte components from cIEF are concentrated over a hundredfold in their separated, narrow zones.
"In column or micro-channel separation technologies, the narrower the component zone is the higher the resolution. Narrow zones in other CE modes can be achieved by injecting a very small 'analyte plug' representing a very small segment of the whole separation channel. Even then, however, component zone broadening is unavoidable during the separation process for the reasons stated above. By contrast, cIEF allows the whole separation channel to be filled with the analyte sample mixture without any deterioration of the separation resolution. In comparison to other CE modes, this provides cIEF with a much higher analyte loading capacity.
"The absence of parabolic flow broadening with static, focused zones and low electropheresis current makes cIEF's separation resolution much less dependent on small dimensional separation channels. The separation channel's cross-sectional dimension can be 2 to 5 times larger than other CE modes with comparable separation resolution. Use of a larger cross-sectional separation channel again further increases sample analyte loading capacity. Higher sample loading provides a higher amount of extracted component material. Increasing the amount of extracted material is highly desirable since it generally increases the analytical success of subsequent analytical techniques such as mass spectrometry.
"For the foregoing reasons, cIEF would appear to be an attractive technique for nano/micro preparative fractionation of closely related proteins from a mixture, for example, variants of hemoglobin arising from mutations to the amino acids sequences; or different forms of recombinant proteins arising from the heterogeneity associated with different post-translational modifications.
"Others have investigated the use of cIEF for fraction collection, as an aspect of CE fractionation generally. For example, Guttman et al [4,5] have studied 'planar' electrophoresis using a capillary cross-connector. By applying different voltage configurations through different reservoirs, a component zone or peak was collected after passing a single-point detector.
"To date, however, others have had limited success in achieving any high degree of precision in the selection and extraction of cIEF component zones, chiefly because of the following limitations to the operation of their devices: 1) mobilization flow is in one direction; 2) mobilization speed is pre-determined and generally fixed and 3) inability to visualize the entire separation zones and detect in real-time any zone-width distortion due to mobilization.
"In conventional 'single point on-column' detection cIEF, the focused zones or peaks within the capillary must be moved, chemically or electroosmotically, past the detection point to be detected. This mobilization step in cIEF requires extra time and distorts the focused peaks, making it difficult to collect pure peaks without cross-contamination when peaks focus in close proximity.
"It has, accordingly, not been possible with existing microfluidic (capillary) electrophoretic devices for micro-preparative fraction collection to observe all of the separated peaks developed by electrophoresis, then select a particular peak of interest and mobilize the entire pattern of peaks, while maintaining or re-establishing the focus of the pattern to bring the selected peak to a separation/collection point.
"It is a principal object of the current invention to provide an integrated micro-scale electrophoresis device (IMED) for the extraction of large (.mu.g) or small (ng) amounts of components (in particular, proteins) separated by cIEF.
"It is a particular object of the invention to provide such an IMED as aforesaid which is adapted for automatic sample injection and capable of high-resolution protein separation, selection of a specific protein zone or peak and precision extraction of the central portion of the selected protein peak ('heart-cut extraction') for further characterization."
As a supplement to the background information on this patent application, VerticalNews correspondents also obtained the inventors' summary information for this patent application: "In its broadest aspect, the apparatus of the invention is apparatus useful for the selective extraction of an analyte from a mixture of analytes using capillary isoelectric focusing, which includes: a capillary separation channel filled with a medium containing a mixture of ionic components in which target analytes migrate are separated into zones and in stationary or near stationary equilibrium; a capillary extraction channel intersecting with and angularly displaced from said capillary separation channel and in fluid communication therewith at the location of intersection; means for causing selected zones of analytes separated by capillary isoelectric focusing to move to said intersection of the separation and extraction channels in a time-independent manner; optical whole column imaging detection apparatus for monitoring the isoelectric focusing process and observing the position of the separated zones of analyte; and means for applying an extraction force to direct a single zone containing a selected analyte into and then out of extraction channel in a time-independent manner.
"According to a preferred embodiment of apparatus according to the invention there is provided a coplanar, cross-channel microfluidic device comprising a sample mixture introduction and separation channel and an extraction channel in fluid communication at a point of intersection; means of producing a pattern of amphoteric components separated into focused, stationary zones within the separation channel; an apparatus for irradiating the whole separation channel with ultraviolet (UV) light and having a detector that can provide UV absorption imaging detection of the whole separation channel providing real-time or at least very, rapid digital images of the cIEF separation process; means of selecting a specific focused, component zone or peak in the separation channel and moving the said peak to the intersection of the separation and extraction channels; the aforementioned whole-channel, real-time imaging detection apparatus to monitor the movement of the selected component peak and to visualize the alignment of said peak to the intersection of the separation and extraction channels; and means for moving the aligned, component peak or a portion of the peak into and then out of the extraction channel for collection or interface to a second analytical apparatus such as a mass spectrometer.
"The invention is also directed to a method for fractionating and extracting analytes capable of resolution by capillary isoelectric focusing, comprising the steps of providing a capillary separation channel and a capillary extraction channel in fluid communication therewith; introducing an analyte sample containing a mixture of ionic analytes prepared for electrophoresis into the separation channel; separating and focusing components of the analyte mixture into separated zones in the separation channel using capillary isoelectric focusing; monitoring the process of capillary isoelectric focusing and positions of the separated analyte zones using whole column imaging detection means; causing the separated zones to move selectively to the intersection of the separation and extraction channels; and applying an extraction force to direct a zone containing desired analytes out through the extraction channel for collection.
"According to particular embodiments of the method of the invention, the separated zones are caused to move along the separation channel to the intersection of the separation and extraction channels by orienting the separation channels so that gravity causes the desired motion. Microfluidic delivery means may be used to move the separated zones and also to apply the extraction force.
BRIEF DESCRIPTION OF THE DRAWINGS
"The invention will now be described further, with reference to the accompanying drawings, in which:
"FIG. 1 is a schematic illustration of essential components of an IMED for use in the separation and extraction of sample components according to the present invention;
"FIG. 2 schematically illustrates an IMED of the same configuration as FIG. 1, but illustrating the arrangement of electrolyte holding tanks and semipermeable membranes used for handling of ampholytes and analyte samples in solution into the IMED;
"FIG. 3 illustrates a variant of the IMED of FIG. 2, having a different configuration of interconnecting separation and extraction channel capillaries;
"FIG. 4 is a graphical representation (electropherograms) of protein zones as light absorbance peaks along the whole length of the separation channel, illustrating the separation, manipulation and extraction of selected component Hemoglobin C by means of the IMED of FIG. 3;
"FIG. 5 presents electropheregrams of absorbance peaks versus separation channel position showing the separation and extraction of selected component Hemoglobin S in the same IMED as employed to produce the results of FIG. 4;
"FIG. 6 shows the cIEF electrophoregram of myoglobin and transferrine using an IMED having the structure shown in FIG. 2;
"FIG. 7 illustrates the mass spectrogram of separated and extracted myoglobin component from a separation carried out by apparatus of the invention and illustrated in FIG. 3; and
"FIG. 8 illustrates the mass spectrogram of separated and extracted transferrine component from the separation carried out by apparatus of FIG. 3."
For additional information on this patent application, see: Wu, Jiaqi; Huang, Tiemin;
Keywords for this news article include: Patents, Peptides, Amino Acids, Hemoglobins, Blood Proteins.
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