Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-09-18
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S029000, C435S377000, C435S378000, C435S396000, C435S397000, C435S402000, C435S287100
Reexamination Certificate
active
06653089
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to systems and methods for selectively treating selected regions of an individual biological cell, and more particularly to systems and techniques utilizing laminar flow channel systems for such treatment.
BACKGROUND OF THE INVENTION
Complex behavior of cells, for example mitosis, growth, movement, metabolism, differentiation, apoptosis, etc. reflect integration of processes occurring in separate micro domains. Investigation of such behaviors require methods for delivering reagents to and/or into cells with subcellular resolution. Currently available techniques now used for micro manipulation of cells, for example micro injection, manipulation using mechanical or optical systems, etc., can, in some instances, provide subcellular resolution, but suffer from various limitations.
For example, micro manipulation techniques, such as the use of optical tweezers (Ashkin, A. and Dziedzic, J. M., “Internal cell manipulation using infrared laser traps,”
Proc. Natl. Acad. Sci. USA,
vol. 86, 7914-7918 (1989), can provide limited subcellular spatial resolution, but such techniques are limited in their molecular specificity. Microinjection techniques can provide molecular specificity; however, they lack spatial control due to the rapid diffusion of small molecules within the cell. In addition, techniques such as microinjection also require physical disruption of the cell plasma membrane in order to provide reagents to the interior of the cell.
Microfluidic systems utilizing a multi-component laminar flow stream have been employed to create microfluidic sensor systems. Such microfluidic sensor systems are described, for example in Weigl, B. H. and Yager, P., “Microfluidic Diffusion-based Separation and Detection,”
Science
283, 346-347 (1999). Kennis et al., “Micro Fabrication Inside Capillaries Using Multi Phase Laminar Flow Patterning”,
Science,
Vol. 285 (1999) describes the use of a laminar flow based microfluidic system for fabricating microstructures in capillaries. Takayama, et al., “Patterning Cells and Their Environment Using Multiple Laminar Fluid Flows and Capillary Networks,”
Proc. Natl. Acad. of Sci. USA,
Vol. 96 (1999) describes using similar laminar flow based microfluidic networks to facilitate the spatial patterning of cells on a substrate and to provide a selected fluid environment to cells attached to a substrate.
Laminar flow occurs when two or more streams having a certain characteristic (low Reynolds number) are joined into a single, multi-component stream, also characterized by a low Reynolds number, such that the components are made to flow parallel to each other without turbulent mixing. The flow of liquids in small capillaries often is laminar. For a discussion of laminar flow and a definitions of the Reynolds number, the reader is referred to any of a large number of treatises and articles related to the art of fluid mechanics, for example, see Kovacs, G. T. A., “Micromachined Transducers Sourcebook,” WCB/McGraw-Hill, Boston (1998); Brody, J. P., Yager, P., Goldstein, R. E. and Austin, R. H., “Biotechnology at Low Reynolds Numbers,:
Biophys. J,
71, 3430-3441 (1996); Vogel, S., “Life in Moving Fluids,” Princeton University, Princeton (1994); and Weigl, B. H. and Yager, P., “Microfluidic Diffusion-based Separation and Detection,”
Science
283, 346-347 (1999), each incorporated herein by reference.
Analytical chemical techniques have utilized laminar flow to control the positioning of fluid streams relative to each other. U.S. Pat. No. 5,716,852 (Yager et al.), describes a chemical sensor including a channel-cell system for detecting the presence and/or measuring the presence of analytes in a sample stream. The system includes a laminar flow channel with two inlets in fluid connection with the laminar flow channel for conducting an indicator stream and a sample stream into the laminar flow channel, respectively. The indicator stream includes an indicator substance to detect the presence of the analyte particles upon contact. The laminar flow channel has a depth sufficiently small to allow laminar flow of the streams and length sufficient to allow particles of the analyte to diffuse into the indicator stream to form a detection area.
U.S. Pat. No. 4,902,629 (Meserol et al.), discusses laminar flow in a description of apparatus for facilitating reaction between an analyte in a sample and a test reagent system. At least one of the sample and test reagent system is a liquid, and is placed in a reservoir, the other being placed in a capillary dimensioned for entry into the reservoir. Entry of the capillary into the reservoir draws, by capillary attraction, the liquid from the reservoir into the capillary to bring the analyte and test reagent system into contact to facilitate reaction.
A variety of references describe small-volume fluid flow for a variety of purposes. U.S. Pat. No. 5,222,808 (Sugarman et al.), describes a capillary mixing device to allow mixing to occur in capillary spaces while avoiding the design constraints imposed by close-fitting, full-volume mixing bars. Mixing is facilitated by exposing magnetic or magnetically inducible particles, within the chamber, to a moving magnetic field.
U.S. Pat. No. 5,300,779 (Hillman et al.), describes a capillary flow device including a chamber, a capillary, and a reagent involved in a system for providing a detectable signal. The device typically calls for the use of capillary force to draw a sample into an internal chamber. A detectable result occurs in relation to the presence of an analyte in the system.
International Patent Publication No. WO 97/33737, published Mar. 15, 1996 by Kim et al., describes modification of surfaces via fluid flow through small channels, including capillary fluid flow. A variety of chemical, biochemical, and physical reactions and depositions are described.
Typical prior art techniques employed for selectively treating single cells or supplying an active substance to the interior of a biological cell are unable to create long-term intracellular gradients, particularly of small molecules (e.g. those with molecular weights less than about 600 and having diffusion coefficients within the cell of more than about 10
−6
cm
2
/s). Microinjection studies and fluorescence recovery after photobleaching (FRAP) studies have shown that such small molecules will diffuse throughout the cytoplasm or myoplasm of a typical mammalian cell attached to a substrate (e.g., an attached mammalian cell having a maximum spread dimension of about 130 &mgr;m) within seconds the intracellular distribution of the molecules will reach 95% of an equilibrium distribution (i.e., there will be no region within the interior of the cell having a concentration of the molecule differing from another region of the cell by more than about 5%) within about 2 to about 5 minutes, even in the presence of some reversible binding of the molecule to immobilized cellular components, which binding tends to decrease the apparent diffusion coefficient (e.g. see Mastro, A. M., Babich, M. A., Taylor, W. D., and Keith, A. D., “Diffusion of small molecules in the cytoplasm of mammalian cells,”
Proc. Natl. Acad. Sci. USA,
Vol. 81, 3414-3418 (1984); and Blatter, L. A. and Wier, W. G., “Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2,”
Biophys. J,
Vol. 58, 1491-1499 (1990)). Thus, such methods are not well suited for creating intracellular gradients of such molecule having long-term duration.
Bradke and Dotti, “The Role of Local Actin Instability in Axon Formation,”
Science
, Vol. 283, 1999, describe a micropipetting technique for selectively treating a region of an axon of a neuron with a cytoskeletal disrupting substance. The technique described utilizes selectively positioned micropipettes to direct a flow of liquid containing the cytoskeletal disrupting substance such that it impinges upon a portion of the axon extending away from the main body portion of the cell. By using this technique, the act
Ingber Donald E.
LeDuc Philip
Naruse Keiji
Ostuni Emanuele
Takayama Shuichi
Ketter James
President and Fellows of Harvard College
Wolf Greenfield & Sacks P.C.
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