Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-10-19
2004-08-31
Noguerola, Alex (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S451000, C204S602000, C204S601000, C204S604000
Reexamination Certificate
active
06783647
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to molecular biology, and in particular, to the use of an applied electric field in a microfluidic system for the manipulation of biological samples comprising cells and cell lysate(s) for subsequent analysis.
BACKGROUND OF THE INVENTION
Interest in microfabricated devices for chemical sensing and analysis has grown substantially over the past decade, primarily because these miniature devices have the potential to provide information rapidly and reliably at low cost. Microchips fabricated on planar substrates are advantageous for manipulating small sample volumes, rapidly processing materials, and integrating sample pretreatment and separation strategies. The ease with which materials can be manipulated and the ability to fabricate structures with interconnecting channels that have essentially no dead volume contribute to the high performance of these devices. See, for example U.S. Pat. Nos. 5,858,195 and 6,001,229, which are commonly owned with this application. To carry out a complete analysis, many different kinds of functional elements can be designed and integrated on microchips. These elements include filters, valves, pumps, mixers, reactors, separators, cytometers and detectors, which can be operatively coupled together under computer control to enable the implementation of a wide range of microchip-based analyses.
One area of particular interest is the analysis of cells and cell populations. At present most techniques for cellular analysis depend upon pooling a population of cells to obtain a large enough quantity of analyte for detection. Pooling of cells, however, obscures any variation in analyte concentration from cell to cell. For many studies average analyte values across a large population of cells will be acceptable; however, for the study of processes such as carcinogenesis, the ability to quantitate analytes in individual cells is required so that rare mutations in cells, which lead to drastic changes in cell metabolism and progression to cancer, can be detected.
With the advances in Capillary Electrophoresis over the last two decades the quantitation of analytes in individual cells has become feasible albeit slow because of the intensive manual manipulations which have to be performed. The potential to automate and integrate cell transport, manipulation and lysis with separation and analyte detection make microfluidic devices a desirable platform for performing high throughput screening of individual cells from large populations. A key step in integrating cell handling with analyte detection and quantitation is providing a method of cell lysis which is rapid and generates small axial extent plugs for subsequent analysis. Because the resolution or separation between any pair of compounds can be detrimentally affected by long injection plug lengths, small axial extent plugs are important for a successful separation. It should be noted, however, that while small axial extent plugs are desired, the contents of the cell after lysis should be spread over several cell volumes so that reaction pathways within the cell are terminated and proteolytic enzymes released from vesicles during lysis are sufficiently diluted. This will prevent the possible digestion of proteins of interest.
The use of an applied electric field for cell lysis is known. For example, the lysis of erythrocytes in suspension by pulsed electric fields has been reported both for bovine (Sale and Hamilton, “Effects of High Electric Fields on Microorganisms III, Lysis of Erythrocytes and Protoplasts”,
Biochim et Biophys Acta,
163:37 (1967)) and human erythrocytes (Kinosita and Tsong, “Voltage-Induced Pore Formation and Hemolysis of Human Erythrocytes”,
Biochim et Biophys Acta,
471:227 (1977); and Kinosita and Tsong, “Hemolysis of Human Erythrocytes by a Transient Electric Filed”,
Proc Nail Acad Sci.,
74:1923 (1977)). These reports indicate that applied electric fields resulting in cellular transmembrane potentials on the order of 1 Volt can result in lysis of erythrocytes. However, these previously reported cell lysis techniques utilizing an electric field are typically carried out in a macroscale device, rather than a microchip device. Consequently, such techniques are lacking in certain respects. Specifically, the conditions employed in macroscale electric cell lysis devices do not consistently release relatively high molecular weight nucleic acid molecules, because such molecules do not readily pass through the pores created in the cell membrane by this lysis technique. Also, the existing macroscale, electric lysis devices function as stand-alone units, thus precluding integration of cell manipulation and lysis with separation and analysis of cell lysate in a unitary device.
Other methods of cell manipulation and/or lysis on microscale devices have been proposed. See, for example, U.S. Pat. Nos. 4,676,274 and 5,304,487. Chemical lysis on a microchip device has been demonstrated by mixing a surfactant, i.e. sodium dodecylsulfate, with canine erythrocyte cells. (Li and Harrison, Anal. Chem., 69:1564-1568 (1997)). This report indicates that the cells were lysed in under 0.3 sec. No subsequent analysis of the cellular contents, however, was reported. Single erythrocytes have been lysed and the cell contents separated using two capillaries across which an electric field is applied. A gap of about 5 &mgr;m is provided between the two capillaries. As intact cells pass through the gap, they are lysed and the contents of the lysed cells are transported to the second capillary for separation. The lysis is presumed to be caused by the mechanical shear stresses induced by the change in electric field strength between the capillaries and the gap region. The gap region is considerably larger in cross sectional area, so that the field strength and, therefore, cell velocity is lower than in the capillaries. See, Chen and Lillard, Anal. Chem., 73:111-18 (2001).
Cell lysis can be considered an extreme form of cell membrane permeablization (poration). Optoporation has been carried out using highly focused light from a pulsed laser (Soughhayer et al., Anal. Chem. 72(6): 1342-1347 (2000)). The laser is focused near the cell at the aqueous/glass interface. When the laser is pulsed, a stress (shock) wave is generated which transiently permeablizes the cell. This technique has been shown to be capable of lysing cells, as well. The occurrence of cell lysis or poration only is a function of the cell's distance from the laser focal spot.
Although the above-mentioned cell lysis techniques of the prior art are useful for certain applications, there exists a need in the art for microchip-based cell manipulations and lysis which is sufficiently rapid to minimize continued cellular activity after lysis, thus producing greater accuracy in the analysis of cell processes.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, there is provided a method of releasing the intracellular contents of at least one cell of a cell-containing fluid sample for analysis. The method of the invention comprises providing a substrate having a microchannel structure which includes one or more microchannel(s). An electric field is generated from a source of electric potential and applied in a spatially defined region of the aforementioned microchannel, which functions as a cell lysis region. The strength of the applied electric field is adequate to induce cell lysis. At least one cell of the fluid sample is positioned in the cell lysis region for a time sufficient to release the intracellular contents into the fluid sample. The released intracellular contents form an analyte plug of narrow axial extent in the microchannel.
According to another aspect of the invention, there is provided a microfluidic system for transport and lysis of at least one cell of a cell-containing fluid sample. The microfluidic system of this embodiment of the invention comprises a source of electric potential and a solid substrate including one or more microchannel(s) with a longitudinal axi
Culbertson Christopher T.
Jacobson Stephen C.
McClain Maxine A.
Ramsey J. Michael
Dann Dorfman Herrell & Skillman P.C.
Noguerola Alex
UT-Battelle LLC
LandOfFree
Microfluidic systems and methods of transport and lysis of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Microfluidic systems and methods of transport and lysis of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Microfluidic systems and methods of transport and lysis of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3273412