Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Reexamination Certificate
1999-01-21
2001-06-12
Le, Que T. (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
C250S235000
Reexamination Certificate
active
06246046
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed generally to non-mechanical scanning techniques and, more specifically, to electronically controlled scanning of micro-area devices such as multi-channel electrophoresis chips, capillary arrays, microtiter plates, matrix hybridization array chips, etc.
2. Description of the Background
Cancer detection and screening utilizing genomic markers has proven very successful for a variety of cancers. These assays typically include amplification of a target sequence followed by separation of the products. Capillary electrophoresis has proven its ability to decrease the separation time requirements over traditional slab gels while maintaining resolution of the DNA fragments for diagnosis. Electrophoretic chips have reduced this separation time requirement even further.
Laser-induced fluorescence detection is a fundamental aspect of the burgeoning chip technology being developed for these and other purposes, and has led to the need for the development of innovative approaches for rapid, sensitive and flexible laser scanning of micro-area devices (i.e. devices having micro-targets) which include such devices as capillary arrays, electrophoresis chips having capillary arrays or point targets, microtiter plates having an array of wells and or point targets, matrix hybridization array chips having a plurality of point targets, and the like.
Capillary electrophoresis (CE) has been demonstrated as a powerful technology for the analysis of DNA, proteins and a variety of small molecules due to its inherent advantages including small sample requirement, high separation speed, and cost-effectiveness. Electrophoretic separations in capillaries have been extrapolated to microfabricated chips where capillary-like channels are etched in the surface of planar sustrates. This development has provided a platform where arrays of microchannels not only compete effectively with slab gels for the parallel processing of multiple samples, but reduce total analysis times by as much as two orders of magnitude. The detection of resolved analyte zones in microchannel electrophoresis is commonly accomplished using laser-induced fluorescence (LIF) detection as a result of its technical simplicity as well as the attomole sensitivity attainable. This is particularly the case with the detection of double stranded DNA which is accomplished via the use of double stranded intercalators, but also with single stranded DNA through covalently-bound fluors. In fact, using a sheath flow cell and a microscope, single molecule detection in a capillary has been demonstrated. The extension of single channel fluorescence detection to multiple channels is analogous to the extension of single detector or array detector or a scanning mode.
One of the most powerful multi-channel detectors available for low-intensity level fluorescence array detection is the charge coupled device or CCD camera. There have been several reports of using CCD cameras for LIF detection in CE. In this particular application, a CCD camera is used to acquire a series of pictures of the fluorescence emission from a point(s) on the capillary as a function of time. Serial analysis of the pictures acquired by the CCD camera allows for molecule movement to be monitored and quantitated. Yeung et al. described a one-color LIF detection system for 96 channel electrophoresis using a CCD camera. “Automation and Integration of Multiplexed On-Line Sample Preparation with Capillary Electrophoresis for High-Throughput DNA Sequencing.” Anal. Chem., 1998, 70, 4044-4053. The most significant advantage associated with detection using a CCD camera is that an increase in the observation zone does not correspond to a decrease in temporal resolution. However, the number of the capillaries or microchannels on a chip is limited by the resolution and sensitivity of the CCD camera. Because the CCD acquires the image through a large amount of stored data, the image processing time for real-time analysis is another obstacle, limiting the number of microchannels that can be effectively utilized on the microchip. Another obvious drawback is the full-field illumination. Because the CCD camera acquires information from all channels simultaneously, full field illumination is required, but this is associated with some disadvantages. First, a long illumination time will increase photodynamic damage and dye bleaching. Second, with all of the channels excitated at the same time, scattering from neighboring channels will effect the detected channel. That is, the channel cross-talk will increase. This second disadvantage will eventually restrict the channel number on a given chip.
Another approach for realizing multi-channel detection is to translate the stage on which the chip is mounted. The stage scanning approach has been demonstrated by Mathies and coworkers using a planar array of capillaries [capillary array electrophoresis (CAE)] mounted on a computer-controlled precision translation stage. Huang et al., “Capillary Array Electrophoresis Using Laser-Excited Confocal Flourescence Detection”, Anal. Chem. 1992, 64, 967-972; Woolley et al., “High-Speed DNA Genotying Using Microfabricated Capillary Array Electrophoresis Chips”, Anal. Chem. 1997, 69, 2181-2186. A laser beam was focused onto the chip by a microscope objective and a portion of the emitted fluorescence was collected by the same objective, followed by confocal detection using a photomultiplier tube (PMT). During electrophoresis, the stage was translated, effectively sweeping the chip back and forth periodically. There is no limitation to the number of the capillaries or multi-channels, but as a result of the mechanical movement with this method, it is difficult to realize ultra-fast scanning rates over wide scanning regions or to scan different regions at the same time, limiting the separation time and real-time processing for electrophoresis.
An alternative approach for realizing multichannel CAE detection is laser beam scanning. There are a number of methods for laser scanning. See Marshall, “Optical Scanning”, Marcel Dekker, Inc., 1991; Trepte et al., “Computer Control for a Galvanometer Scanner in Confocal Scanning Laser Microscope.” Optical Eng. 1994, 33, 3774-3780. Most of the commercially-available scanning microscopes are based on mechanically movable mirrors positioned by motors. The obvious advantage associated with reflection scanning is that it is wavelength-independent. However, due to its mechanically-controlled nature, it is difficult to achieve ultrahigh scanning rates and cancel out the mechanical noise, e.g., wobble. This can result in the distortion in a scanned image or signal and, with capillaries or multi-channels, can be associated with defocusing of the laser beam during detection. Mathies group has recently presented a circular 96-channel silica chip with LIF detection achieved via a spinning objective system exploiting a similar galvanometeric approach to that described previously. Frederick Conference on Capillary Electrophoresis, 1998, Frederick, Md.
Electro-optical scanning is based on the fact that the optical properties of certain materials have an electric field dependence (E-O effect). An advantage of the electro-optical scanner is that, with most applications, it can be treated as a capacitor with the speed of operation dependent on the output characteristics of the drive circuit. Consequently, nanosecond sweep rates can be obtained. Unfortunately, as yet, few materials have been discovered that exhibit a strong enough E-O effect to make them useful as practicable E-O devices. In addition, the commercially-available devices are physically too large to be applied to microscopy.
Acousto-optic devices appear to be a relatively unexplored approach to laser beam scanning for excitation of fluorescence targets on micro-area devices. AOD's utilize the diffraction effect induced by optical gratings to achieve laser beam deflection. The acousto-optic (A-O) effect occurs when a light beam passes through a trans
Huang Zhili
Huhmer Andreas
Landers James P.
Le Que T.
Thorp Reed & Armstrong
University of Pittsburgh
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