Radiant energy – Luminophor irradiation
Reexamination Certificate
2002-05-15
2003-09-02
Sugarman, Scott J. (Department: 2873)
Radiant energy
Luminophor irradiation
C250S461100
Reexamination Certificate
active
06614030
ABSTRACT:
TECHNICAL FIELD
The field of this invention is fluorescent detection in microfluidic arrays.
1. Background
The combination of combinatorial chemistry, sequencing of the genomes of new species and relationships between genotype and physical and biological traits has greatly expanded the need to perform determinations of different events The multiplicity of new compounds that can be prepared using various forms of combinatorial chemistry and the numerous targets involving wild-type and mutated genes, had extraordinarily increased the number of determinations of interest in developing compounds having biological activity. These compounds include drugs, biocides, pesticide resistance, disease organism resistance and the like. In addition, the interest in discriminating between different genomes, relating specific mutations to phenotypes, defining susceptibilities to various environmental effects in relation to single nucleotide polymorphisms, and identifying the genomes of organisms to provide better defenses against the organisms has expanded the need for rapid inexpensive devices and methodologies for carrying out these and other determinations.
Recently, microfluidic arrays have been developed which allow for a multiplicity of reservoirs and channels to be associated with a small card or chip, where by using high voltages, various operations can be performed. The arrays provide for individual networks, which exist in combination on a single chip, so that a plurality of determinations may be performed concurrently and/or consecutively By having channels that have cross-sections in the range of about 500 to 5000 &mgr;
2
, operations can be carried out with very small volumes In addition, by having very sensitive detection systems, very low concentration of a detectable label may be employed This allows for the use of very small samples and small amounts of reagents, which have become increasingly more sophisticated and expensive Micofluidic arrays offer the promise of more rapid throughput, increasingly smaller times to a determination and increasingly smaller amounts of sample and reagents being required
The use of microfluidic arrays, however, is not without its challenges. The microfluidic arrays are desirably made in molded plastic, so as to provide a reduced cost of the chip. By molding the chip and providing for ridges on a mold to form the channels, the channels may not run true and may be displaced from their proper positions, as well as being slightly curved rather than perfectly straight. In addition, the plastic frequently autofluoresces. Since, the frequently used label is a fluorescent label, the signal from the label must be able to be distinguished from the autofluorescent signal. There is the problem of how to obtain a reliable fluorescent signal, in effect compromising maximizing the signal from the detectable label while minimizing the background signal.
In addition, the channel walls are not orthogonal to the cover plate, so that the depth of the irradiation will vary, depending upon the site of entry of the excitation beam into the channel. Where the excitation beam encounters the wall, the signal is degraded due to the reduced number of fluorophores which are excited and the excitation of the fluorophores in the wall. Therefore, precise positioning of the excitation beam in the channel is necessary for reproducible and accurate results.
2. Brief Description of Related Art
A number of patents have been published describing systems for detecting fluorescent signals in capillary arrays, such as U.S. Pat. Nos. 5,296,703 and 5,730,850, as well as WO98/49543.
SUMMARY OF THE INVENTION
An optical fluorescence detection system is provided for use with microfluidic arrays. The detection and orientation system comprises an optical train for receiving and processing light from a source of light and directing the light onto a microfluidic channel in a solid substrate. The optical train is moved across the surface of the solid substrate, crossing the channel and receiving the light emanating from the solid substrate. The optical train directs and processes the light from the solid substrate surface and directs the light to a detector. The signal from the detector is received by a data analyzer, which analyzes the signals and directs the optical train to the center of the channel in relation to the observed signals from the bulk material of the solid substrate, the edges of the channel and from the channel. Fluorescent components in the channel are detected by the fluorescence produced by the excitation light, where the emitted light is processed by the optical train and analyzed for the presence of fluorescence in the channel resulting from the fluorescent components in the channel, correcting for any fluorescence from the solid substrate.
The optical fluorescence detection system employs a plurality of miniaturized confocal microscope systems aligned in orientation with a plurality of channels of a microfluidic array. The systems are mounted on a movable support for alignment with sets of channels. The supports may be mounted on a carriage for alignment with different sets of channels. An irradiation unit comprises a source of light and processing means, such as lenses, dichroic mirrors, filters, gratings or the like, to reject light outside the wavelength range of interest. A single light source may be used and the beam split into a plurality of optical fibers for individual distribution of beamlets for channel irradiation. Similarly, the individual signals from each of the channels is directed by individual optical fibers to a common detector. Alternatively, individual light sources may be used for each confocal microscope system, such as LEDs or laser diodes.
The methodology allows for accurate, reproducible determination of a fluorescent signal from each of the channels. In order to achieve the desired sensitivity for detection, the center of each channel is determined, either when the channel is empty (air) or when a liquid is present, usually containing a fluorescent dye. Depending upon the degree of autofluorescence of the microfluidic array substrate, the optical system may look at fluorescent light, where there is sufficient autofluorescence to provide a detectable signal or scattered light, usually where the autofluorescence is low. In the case of scattered light, one would be detecting a different wavelength from the light, which would result from autofluorescence.
There are two different forms of delivering excitation: single mode fiber delivery or no fiber, where a laser and splitting must be done by discrete mirrors or a diffraction optical element; or multi-mode fiber delivery, where either a lamp or a laser may be used and splitting is done by homogenizing the laser or lamp light and then splitting using a multi-mode fiber array. The source of light will usually be a laser, generally being a laser producing a light beam having a wavelength in the range of about 250 to 800 nm, usually 488 nm, 532 nm or 633 nm.
Depending upon the source of light, such as a laser, a filter may be used to attenuate the intensity of the light to minimize photobleaching and photodestruction of the fluorescent labels. The light is then split into a plurality of rays or beamlets by a diffractive optical element, a combination of beam splitter elements, such as discrete mirrors, or other means, such as discrete beam splitters and fiber optic arrays. Each of the resulting beams is then directed to the individual confocal microscope associated with the channels. Either a single mode or multimode fiber may be employed, where one may use a multimode fiber optic array to split the illumination into N beamlets, where N is the number of optical trains to be illuminated. The fiber will generally have a diameter in the range of about 25 to 75 &mgr;m, particularly about 50 &mgr;m and a length in the range of about 1 to 1000 mm.
The confocal housing can be very compact, where the portion enclosing the optical train, usually in conjunction with other enclosed areas associated with the optical
Bjornson Torleif Ove
Maher Kevin
Smith Timothy F.
Aclara Biosciences, Inc.
Hanig Richard
Macevicz Stephen C.
Sugarman Scott J.
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