Method of analyzing multiple sample simultaneously by...

Details Method of analyzing multiple sample simultaneously by... Method of analyzing multiple sample simultaneously by...

2002-06-05

2004-09-07

Rosenberger, Richard A. (Department: 2877)

Optics: measuring and testing

For light transmission or absorption

Of fluent material

C356S440000

Reexamination Certificate

active

06788414

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to a method of analyzing multiple samples simultaneously by detecting absorption and systems for use in such a method.
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 have become the 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 material, it will be 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. With attractive features such as rapid analysis time, high separation efficiency, small sample size, and low solvent consumption, CE is being increasingly used as an alternative or complementary 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 loss of the ability (inherent in slab gels) to accommodate multiple lanes in a single run. Highly multiplexed capillary electrophoresis, by making possible hundreds or even thousands of parallel sequencing runs, represents an attractive approach to overcoming 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, light-induced fluorescence is exclusively employed as the detection method.
While fluorescence detection is suitable for DNA sequencing applications because of its high sensitivity and special labeling protocols, UV absorption detection has remained very useful because of its ease of imnplementation and wide applicability, especially for the deep-UV (200-220 nm) detection of organic and biologically important compounds. A capillary isoelectric focusing system using a two-dimensional CCD detector, in which one dimension represents the capillary length and the other dimension records the absorption spectrum, has been described by Wu and Pawliszyn,
Analyst
(
Cambridge
), 120, 1567-1571 (1995). The system has been used for 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 litnited 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 scheme utilized is not suitable for densely packed capillary arrays because of the presence of mechanical slits to reict the light paths. Further, in order to avoid cross-talk, only square capillaries 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 the flow stream is dispersed by a grating and recorded across the linear array. A capillary zone electrophoresis system using a photodiode array as the imaging absorption detector has been described by Culbertson and Jorgenson,
Anal. Chem
., 7, 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 million electrons), it is superior to the CCD for absorption detection. Time-correlated integration is applied to improve the signal-to-noise ratio (S/N).
What is still needed is an absorption detection approach for the simultaneous analysis of multiple systems. One such system is shown in U.S. Pat. No. 5,900,934 (Gilby et al.). 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 disposed 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 a respective photosensitive element of the photodetector array to effectt masuremcnt 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., capillaries, is difficult for several reasons. For example, positioning the capillaries with equal separation there between is difficult as the capillaries generally are not of equal dimension, e.g., diameter tolerances vary greatly. Further, for example, the mask geometry does not provide identical light paths, which leads to nonlinear response. Also, a mask can produce stray light, which leads to poor detection limits, and does not completely eliminate crosstalk from the adjacent capillaries, since the light beams are diverging and cannot escape the detector element. In addition, a mask can be difficult to manufacture, due to the requirement of uniformity. Also, Gilby places the sample and the PDA too close together, resulting in stray light, cross talk and the inability to use the maximum pathlength of light.
Thus, in view of the disadvantages inherent to the methods and systems in the art, there remains a need a method of analyzing multiple samples simultaneously by absorption detection. It is an object of the present invention to provide such a method. It is another object of the present invention to provide a system for use in such a method. These and other objects and advantages of the present invention as well as additional inventive features will become apparent to one of ordinary skill in the art from the detailed description provided herein.
The present invention also addresses other disadvantages in the art. For example, since the invention of the polymerase chain reaction (PCR) in 1985 by Kary Mullis, the uiate in sensitivity, together with increasing ease in implementation, have placed this technique in a central position i

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