Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-04-10
2003-07-01
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C422S051000, C422S063000, C422S093000, C422S098000, C422S105000, C435S007400, C435S007600, C435S287100
Reexamination Certificate
active
06586253
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to cell assays on microchips.
BACKGROUND OF THE INVENTION
Several methods are known for enzymatic assays, such as use of flow cytometry, fluorescence microscopy, and capillary electrophoresis, and a great deal of work is being done on these systems to improve system performance characteristics such as average activity, distribution, separation of products, sensitivity, throughput, analysis time and quantitation. Due to the very small quantities involved, and the desire to detect small concentrations of products, improvement of all of the system performance characteristics, without degradation of any, is very difficult. There is thus a need for a device system showing improvement in all of these system performance characteristics.
One particular enzyme for which detection of small quantities is important is &bgr;-galactosidase. Deficiency in &bgr;-galactosidase is symptomatic of diseases such as GM1-gangliosidosis, galactosialidosis and Morquio B syndrom (mucopolysaccharidosis (MPS) IV B). These are genetic diseases that onset at various ages, and affect a variety of cell types, including brain tissue, leukocytes and skin fibroblasts. &bgr;-galactosidase is also used as a product of a reporter gene in recombinant DNA work. Enzymatic assays are available in which suspended cell populations from a patient are analyzed using flow cytometry. While this is a single cell method, the technique reports the results from an ensemble of cells measured at one point in time.
SUMMARY OF THE INVENTION
This invention relates to a microchip system in which products from lysed cells are detected. A microchip format allows for detailed evaluation of the kinetics of response of each cell in an enzymatic assay. This makes it is easier to sort cell responses into different categories. In flow cytometry the cells remain intact and the substrate must cross the cell membrane. The limited amount which transports across, and the low rate at which this occurs means that cells must be incubated for hours before analysis. Performing the &bgr;-galactosidase assay within a microchip allows for a novel sequnce of analysis that substantially speeds up the assay over flow cytometry methods.
There is therefore provided in accordance with an aspect of the invention, a method for the detection of cell contents, the method comprising the steps of introducing a cell into a channel in a microchip; lysing the cell to release cell contents into the channel; moving the cell contents towards a detection zone; and detecting the cell contents at the detection zone.
In accordance with a further aspect of the invention, there is provided an apparatus for the detection of cell contents, the apparatus comprising: a microchip; a cell mobilization channel formed in the microchip, the cell mobilization channel having a cell introduction end and a detection end; a cell mobilizer operably connected with the cell introduction end for moving cells from the cell introduction end to the detection end; means for lysing cells in the cell mobilization channel at a lysing zone, the lysing zone being located between the cell introduction end and the detection end; and a detector, disposed adjacent the detector end, arranged to detect cell contents appearing at the detector end that have been moved from the lysing zone to the detector end by the cell mobilizer.
In further aspects of the invention, the cells are mammalian cells and the cell contents comprise &bgr;-galactosidase. The cell contents may be moved towards the detection zone by electrophoretic, electroosmotic forces, pumping, or other mobilization methods. The cell may be lysed by various techniques such as application of an electric field across the cell, or by introduction of a reagent into the channel. The channel dimensions should be similar to the cell dimensions, preferably not more than about twelve cell diameters.
These and other aspects of the invention are described in the detailed description of the invention and claimed in the claims that follow.
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Andersson Per E.
Arriaga Edgar
Harrison D. Jed
Ocvirk Gregor
Christensen O'Connor Johnson & Kindness PLLC
Nguyen Bao-Thuy L.
The Governors of the University of Alberta
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