Chemistry: analytical and immunological testing – Measurement includes change in volume or pressure
Reexamination Certificate
2000-05-11
2003-01-14
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Measurement includes change in volume or pressure
C436S034000, C436S052000, C436S180000, C436S518000, C422S050000, C435S091100
Reexamination Certificate
active
06506609
ABSTRACT:
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
A variety of cell-based assays are of considerable commercial relevance in screening for modulators of cell-based activity. For example, compounds which affect cell death can have profound biological activities and are desirably screened for in cell-based assays. Cell death has become recognized as a physiological process important in normal development, hormonal regulation of various tissues, and, e.g., in regulation of the receptor repertoires of both T and B lymphocytes. The finding that a pattern of morphological changes is common to many examples of programmed cell death (or PCD) led to the suggestion of a common mechanism, and the term “apoptosis” was defined to include both the morphological features and the mechanism common to such programmed cell death (Kerr et al.,
Br. J. Cancer
26:239). This concept was extended by the finding that nuclear DNA fragmentation correlates well with apoptotic morphology (Arends et al.,
Am. J. Pathol.
136:593 (1990)), and the scientific literature contains many examples of PCD accompanied by these features. There are also clear examples of PCD in the absence of apoptotic morphology or DNA fragmentation (Clarke,
Anat. Embryl.
181:195 (1990), Martin et al,
J. Cell Biol.
106:829 (1988), and Ishigami et al.,
J. Immunol.
148:360 (1992)).
Cell-based assay systems model relevant biological phenomena, and have generally been widely adopted as screening assays, e.g., when screening for a compound's effect(s) on apoptosis or other biological phenomena. Pioneering technology providing cell- and other particle-based microscale assays are set forth in Parce et al. “High Throughput Screening Assay Systems in Microscale Fluidic Devices” WO 98/00231; in PCT/US00/04522, filed Feb. 22, 2000, entitled “Manipulation of Microparticles In Microfluidic Systems,” by Mehta et al.; and in PCTUS00/04486, filed Feb. 22, 2000, entitled “Devices and Systems for Sequencing by Synthesis,” by Mehta et al.
Other cell-based assays include various methods for the preparative or analytic sorting of different types of cells. For example, cell panning generally involves attaching an appropriate antibody or other cell-specific reagent to a solid support and then exposing the solid support to a heterogeneous cell sample. Cells possessing, e.g., the corresponding membrane-bound antigen will bind to the support, leaving those lacking the appropriate antigenic determinant to be washed away. Other well-known sorting methods include those using fluorescence-activated cell sorters (“FACSs”). FACSs for use in sorting cells and certain subcellular components such as molecules of DNA have been proposed in, e.g., Fu, A. Y. et al. (1999) “A Microfabricated Fluorescence-Activated Cell Sorter,”
Nat. Biotechnol.
17:1109-1111; Unger, M., et al. (1999) “Single Molecule Fluorescence Observed with Mercury Lamp Ilumination,”
Biotechniques
27:1008-1013; and Chou, H. P. et al. (1999) “A Microfabricated Device for Sizing and Sorting DNA Molecules,”
Proc. Nat'l. Acad. Sci.
96:11-13. These sorting techniques utilizing generally involve focusing cells or other particles by flow channel geometry.
While cell-based assays are generally preferred in certain microscale screening applications, certain of these assays are difficult to adapt to conventional notions of high-throughput or ultra high-throughput screening assay systems. For example, one difficulty in flowing assay systems is that, during pressure-based flow of fluids in channels, non-uniform flow velocities are experienced. Faster fluid and material flow is observed in the center of a moving fluid stream than on the edge of a moving fluid stream. This non-uniform flow velocity reduces throughput for flowing assays, because assay runs have to be spaced well apart in the fluid stream to prevent overlap of materials moving at different velocities.
Accordingly, it would be advantageous to provide mechanisms for facilitating cell-based assays, including cell sorting techniques, especially in microscale systems. Additional microscale assays directed at subcellular components, such as nucleic acids would also be desirable. The present invention provides these and other features which will become clear upon consideration of the following.
SUMMARY OF THE INVENTION
The present invention relates to methods of focusing particles in microchannels, e.g., to improve assay throughput, to sort particles, to count particles, or the like. In the methods of the invention, cells and other particles are focused in the center of, to one side of, or in other selected regions of microscale channels, thereby avoiding, e.g., the above noted difficulties inherent in pressure-based flow of particles. Furthermore, the device structures of the present invention are optionally integrated with other microfluidic systems. Other reactions or manipulations involving cells, other particles, or fluids upstream of the detection zone are also optionally performed, e.g., monitoring drug interactions with cells or other particles.
In one aspect, the invention provides methods of providing substantially uniform flow velocity to particles flowing in a first microchannel. In the methods, the particles are optionally flowed in the microchannel, e.g., using pressure-based flow, in which the particles flow with a substantially non-uniform flow velocity. Prior to performing the flowing step, the particles are optionally sampled with at least one capillary element, e.g., by dipping the capillary element into a well containing the particles on a microwell plate and drawing the particles into, e.g., reservoirs, microchannels, or other chambers of the device. The particles (e.g., a cell, a set of cells, a microbead, a set of microbeads, a functionalized microbead, a set of functionalized microbeads, a molecule, a set of molecules, etc.) are optionally focused horizontally and/or vertically in the first microchannel to provide substantially uniform flow velocity to the particles in the first microchannel. Particles are optionally focused using one or more fluid direction components (e.g., a fluid pressure force modulator an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like). Additional options include sorting, detecting or otherwise manipulating the focused particles.
The particles are horizontally focused in the microchannel, e.g., by introducing a low density fluid and a high density fluid into the microchannel, causing the particles to be focused in an intermediate density fluid present between the high density fluid and the low density fluid. The particles are also optionally focused in a top or a bottom portion of the microchannel by introducing a high or a low density fluid into the microchannel with the flowing particles. The particles are vertically or horizontally focused in the microchannel, e.g., by simultaneously introducing fluid flow from two opposing microchannels into the first microchannel during flow of the particles in the first channel. Vertical focusing is also optionally achieved to one side of a microchannel by simultaneously introducing fluid flow from, e.g., a second microchannel into the first microchannel during flow of the particles in the first microchannel.
In another aspect, the invention also provides particle washing or exchange techniques. For example, focused cells or other particles are optionally washed free of diffusible material by introducing a diluent into the first microchannel from at least a second channel and removing the resulting diluted diffused product comprising diluent mixed with the diffusible material through at least a third microchannel.
A
Alajoki Marja Liisa
Chow Andrea W.
Dubrow Robert S.
Kopf-Sill Anne R.
Parce J. Wallace
Caliper Technologies Corp.
Filler Andrew L.
Sines Brian
Warden Jill
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