Optics: measuring and testing – By electrophoresis
Reexamination Certificate
2002-10-11
2004-12-21
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
By electrophoresis
Reexamination Certificate
active
06833919
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to a multiplexed, absorbance-based capillary electrophoresis system and to a method of using it.
BACKGROUND OF THE INVENTION
The rapid development of biological and pharmaceutical technology has posed a challenge for high-throughput analytical methods. For example, current development of combinatorial chemistry has made it possible to synthesize hundreds or even thousands of compounds per day in one batch. Characterization and analysis of such huge numbers of compounds has created a bottleneck. Parallel processing (i.e., simultaneous multi-sample analysis) is a natural way to increase the throughput. However, due to limitations related to column size, pressure requirements, detector and stationary phase materials, it is very difficult to build a highly multiplexed high-performance liquid chromatography (HPLC) system. The same goes for building a highly multiplexed gas chromatography (GC) system.
High performance capillary electrophoresis (CE) has rapidly become an important analytical tool for the separation of a large variety of compounds ranging from small inorganic ions to large biological molecules. To perform a conventional separation, a capillary tube is filled with a buffer solution, a sample is loaded into one end of the capillary tube, both ends of the capillary tube are immersed in the buffer solution and a large potential is applied across the capillary tube. The sample components are separated electrophoretically as they migrate through the capillary tube.
CE is used for general separations, enantiomeric separations, the peptide mapping of proteins, amino acid analysis, nucleic acid fractionation and the quantitative measurement of acid dissociation constants (pK
a
values) and octanol-water partition coefficients (log P
ow
values). What all these applications have in common is the measurement of the mobility of chemical species in a capillary tube.
With attractive features such as rapid analysis time, high separation efficiency, small sample size, and low solvent consumption, CE is increasingly used as an alternative or complimentary technique to HPLC. For example, the use of capillary gel electrophoresis has greatly improved DNA sequencing rates compared to conventional slab gel electrophoresis. Part of the improvement in speed, however, has been offset by the inability to accomodate multiple lanes in a single run that is inherent in slab gels. Highly multiplexed capillary electrophoresis, by making possible hundreds or even thousands of parallel sequencing runs, represents an attractive approach to overcome the current throughput limitations of existing DNA sequencing instrumentation. Such a system has been disclosed in U.S. Pat. Nos. 5,582,705 (Yeung et al.), 5,695,626 (Yeung et al.) and 5,741,411 (Yeung et al.). In this system, a fluorescent sample is separated by electrophoresis inside a capillary tube. A laser irradiates one section of the capillary tube. When the sample component migrates through the irradiated portion of the tube, the fluorescence emission is detected by an optical detector.
While fluorescence detection is suitable for DNA sequencing applications because of its high sensitivity and special labeling protocols, many samples of interest do not fluoresce. UV absorption detection is useful because of its ease of implementation and wider applicability, especially for the deep-UV (200-220 nm) detection of organic and biologically important compounds. In a UV detection system, a section of capillary tube is irradiated with a UV light source. A photodetector detects the light that passes through the tube. When a UV absorbing sample component passes through the irradiated portion of the capillary tube, the photodetector detects less passed light (indicating absorbance). In this way an electropherogram, a plot of absorbance versus time, can be produced.
A capillary isoelectric focusing system using a two-dimensional charge-coupled device (CCD) detector, in which one dimension represents the capillary length and the other dimension records the absorbance spectrum, has been described by Wu and Pawliszyn, Analyst (Cambridge), 120, 1567-1571 (1995). The system has been used with two capillary tubes, but is not easily adapted for three or more capillary tubes because the system requires the capillary tubes to be separated by space. Instead of providing wavelength resolution in the second CCD dimension, isoelectric focusing in two capillary tubes is simultaneously monitored. The use of optical fibers for illumination, however, has led to low light intensities and poor UV transmission. So, only visible wavelengths have been employed for the detection of certain proteins. Because the CCD has a very small electron well capacity (about 0.3 million electrons), the limit of detection (LOD) of this system is limited by the high shot noise in absorption detection. The use of the CCD produces an overwhelming amount of data per exposure, limiting the data rate to one frame every 15 seconds. Also, the imaging scan utilized is not suitable for densely packed capillary arrays because of the presence of mechanical slits to restrict the light paths. Further, in order to avoid cross-talk, only square capillary tubes can be used.
Photodiode arrays (PDA) are used in many commercial CE and HPLC systems for providing absorption spectra of the analytes in real time. Transmitted light from a single point in a flow stream is dispersed by a grating and recorded across a linear array. A capillary zone electrophoresis system using a photodiode array as an imaging absorption detector has been described by Culbertson and Jorgenson, Anal. Chem., 70, 2629-2638 (1998). Different elements in the array are used to image different axial locations in one capillary tube to follow the progress of the separation. Because the PDA has a much larger electron well capacity (tens of millions of electrons), it is superior to the CCD for absorption detection. Time-correlated integration is applied to improve the signal-to-noise ratio (S/N).
Gilby described an absorption detection approach for the simultaneous analysis of multiple systems in U.S. Pat. No. 5,900,934. This system includes a photodetector array comprising a plurality of photosensitive elements connected to provide a serial output. The elements are typically pixels of a photodiode array (PDA). The elements are illuminated by a light source positioned to illuminate at least a portion of the photodetector array. The light source may be an AC or DC mercury lamp or other useable light source for chromatography. An array of separation channels is disclosed between the light source and the photodetector array, each of the separation channels having a lumen, a sample introduction end and a detection region disposed opposite the sample introduction end. The array is a multiple parallel capillary electrophoresis system. A mask element having at least one aperture for each associated separation channel is required. Each aperture corresponds to its associated separation channel, thereby selectively permitting light from the light source to pass through the lumen of its associated separation channel. At least a portion of the light passing through the lumen of the associated separation channel falls on the respective photo sensitive element of the photo detector array to effect measurement of absorption of light by a sample introduced into the sample introduction end of the associated separation channel.
The system described by Gilby et al. has disadvantages because it limits the amount of light impinging on the separation channel, providing less than desirable light intensity to the PDA. Further, aligning the apertures and the mask elements with the separation channels, e.g., capillary tubes, is difficult for several reasons. For example, positioning the capillary tubes with equal separation there between is difficult as the capillary tubes are generally not of equal dimension, e.g., diameter tolerances very greatly. Further, for example, the mask geometry does not provide identical light pat
Kenseth Jeremy R.
Pang Ho-Ming
Strasburg Roy F.
COMBISEP
McKee Voorhees & Sease, P.L.C.
Stafira Michael P.
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