초록 ▼

The invention provides a device, system and method that enables a microfluidic flow lysometer cell analyzer. Using a population of suspended living cells. cell surface molecule detection reagents, and cell cytoplasm (or nuclear) molecule detection reagents, this microfluidic cell analyzer can rapidl

The invention provides a device, system and method that enables a microfluidic flow lysometer cell analyzer. Using a population of suspended living cells. cell surface molecule detection reagents, and cell cytoplasm (or nuclear) molecule detection reagents, this microfluidic cell analyzer can rapidly analyze a population of cells by running them on a one-at-a-time basis through small capillary channels. The cell's morphology or surface markers are analyzed, then the cells are lysed, and the molecules present in the cell's cytoplasm or nuclear material are analyzed. Cell morphology is then analyzed as the cell surface molecules are correlated with the molecules present in the same cell's cytoplasm or nucleic acids, and this correlated cell population data is then presented to a user for interpretation. The invention also addresses issues such as device fouling, correction for experimental artifacts (incomplete cell lyis, variable cell debris volume effects), and inadequate data collection that hampered earlier efforts in this area.

대표청구항 ▼

1. A method for performing a population analysis on a plurality of cells while in transit in at least one dynamic microfluidic pathway, the method comprising: providing a population of cells having cell surface indicia;transmitting the population of cells through the microfluidic pathway;analyzing t

1. A method for performing a population analysis on a plurality of cells while in transit in at least one dynamic microfluidic pathway, the method comprising: providing a population of cells having cell surface indicia;transmitting the population of cells through the microfluidic pathway;analyzing the cell surface indicia to obtain a first set of data corresponding to each cell of the population of cells;utilizing a processor to compare the first set of data with at least one preset user criteria to identify at least one cell satisfying the preset user criteria;utilizing the processor to control operation of a cell lyser to selectively lyse the at least one cell while it transits a microfluidic pathway to produce cell debris that exposes cell interior indicia, wherein cells that do not satisfy the preset user criteria are not lysed; andanalyzing the cell interior indicia for at least one of a molecule and a cellular component. 2. The method according to claim 1, in which the cell surface indicia are selected from the group consisting of cell size, cell shape, cell morphology, cell narrow angle light scattering, cell wide angle light scattering, cell electrical characteristics, cell surface membrane molecules, and cell surface markers. 3. The method according to claim 1, in which the cell interior indicia are selected from the group consisting of cell cytoplasmic-side membrane molecules, cell cytoplasm molecules, cell nuclear membrane molecules, cell nuclear molecules, DNA, RNA, cell interior proteins, cell interior lipids, cell interior carbohydrates, cell interior cofactors, cell interior ions, cell interior ATP, cell organelles, and cell organelle molecules. 4. The method according to claim 1, further comprising: combining data from at least one of the cell's surface indicia and data from at least one of the cell's interior indicia from a plurality of the at least one cell to produce a population analysis of the cells. 5. The method according to claim 1, wherein at least one of the cell surface indicia and the cell interior indicia is analyzed while the at least one cell transits the microfluidic pathway, and wherein the at least one cell is alive as it enters the microfluidic pathway. 6. A method for performing a population analysis on a plurality of cells, in which the plurality of cells is analyzed on an individual cell-by-cell basis for specific cell surface markers or specific morphological characteristics, and the individual data obtained on a cell-by-cell basis then assembled into a statistical analysis of the population of cells, the method comprising: providing a population of cells;causing the population of cells to travel through a microfluidic pathway;analyzing the population of cells to obtain a first set of data corresponding to each cell of the population of cells, the first set of data describing at least one of cell surface markers and specific morphological characteristics identify at least one cell having predetermined morphological characteristics;utilizing a processor to compare the first set of data with at least one preset user criteria to identify at least one cell satisfying the preset user criteria;utilizing the processor to control operation of a cell lyser to selectively lyse the at least one cell; andanalyzing the at least one lysed cell. 7. The method of claim 6, wherein the step of lysing the at least one cell is selected from the group consisting of electrical lysing, chemical lysing, osmotic lysing, ultrasonic lysing, laser lysing, and heat lysing methods. 8. The method of claim 6, in which the step of analyzing the population of cells to identify at least one cell having predetermined morphological characteristics is selected from the group consisting of electrode-based electronic measuring methods, light scattering methods, fluorescence detection methods, luminescence detection methods, light absorbance detection methods, automated video microscopy, and vision recognition methods. 9. The method of claim 6, wherein the step of causing the population of cells to travel through a microfluidic pathway utilizes a moving fluid stream either with or without a sheath fluid, and is capable of transporting the cells through at least part of the microfluidic pathway in an intact state; and wherein the step of lysing the at least one cell occurs while the at least one cell is transiting through the microfluidic pathway. 10. The method of claim 6, in which the step of analyzing the at least one lysed cell is a biochemical analysis method selected from the group consisting of fluorescent ion indicator methods, luminescent ion indicator methods, chromogenic enzyme substrate methods, fluorescent enzyme substrate methods, luminescent enzyme substrate methods, fluorescent antibody methods, enzyme labeled antibody methods, luminescent antibody methods, molecular beacon methods, genetic analysis device methods, fluorescent nucleic acids methods, and luminescent nucleic acid methods. 11. The method of claim 6, in which the method of analyzing the at least one lysed cell further comprises compensating for the distorting effects caused by the variable volume of the cell lysis field by: incorporating a fluorescent or luminescent tracking dye into the cells before lysis; and monitoring the fluorescence or luminescence distribution of the tracking dye when it is released from the at least one lysed cell. 12. The method of claim 6, further comprising using a processor to analyze the population of cells to identify at least one cell having predetermined morphological characteristics. 13. The method of claim 6, wherein analyzing the population of cells to identify at least one cell having predetermined morphological characteristics comprises using a processor to analyze at least one of cell surface markers and cell morphological characteristics, wherein the analysis comprises: obtaining a first set of data pertaining to at least one of the cell surface markers and the cell morphological characteristics of the at least one cell;biochemically analyzing at least one of the cytoplasm and nuclear debris field of the at least one cell;obtaining a second set of data pertaining to at least one of the cytoplasm and the nuclear debris field; andcorrelating the first set of data and the second set of data according to user determined criteria, the results of which are at least one of stored and transmitted. 14. A device for correlating an individual cell's cell surface markers or cell morphological characteristics with one or more molecules present in the cell cytoplasm or nucleus of the individual cells; the device being capable of operating on a plurality of individual cells selected from a population of cells; comprising: one or more microfluidic pathways containing a moving fluid stream capable of transporting the individual cells through at least a portion of the microfludic pathways in an intact state;at least one transit analyzer configured to analyze the cell surface markers or the cell morphological characteristics of the individual cells while the cells are transiting the microfluidic pathways in an intact state;a cell lyser configured to lyse the individual cells while the cells are transiting the microfluidic pathways to expose a cytoplasmic and nuclear debris field; andat least one biochemistry analyzer configured to biochemically analyze the cytoplasmic or nuclear debris field of the lysed cells for the one or more molecules present in the cell cytoplasm or the nucleus of the individual cell. 15. The device of claim 14, in which the cell lyser is selected from the group consisting of electrical lysers, chemical lysers, osmotic lysers, ultrasonic lysers, laser lysers, and heat lysers. 16. The device of claim 14, in which the transit analyzer used to analyze the cell surface markers or the cell morphological characteristics is selected from the group consisting of electrode based electronic sensors, light scattering sensors, color detection sensors, fluorescence detection sensors, luminescence detection sensors, and automated video microscopy vision recognition sensors. 17. The device of claim 14, in which the microfluidic pathways of the device carry the cells in a moving fluid stream that is protected from contact with at least one wall of the microfluidic pathways by a sheath fluid. 18. The device of claim 14, in which the biochemistry analyzer configured to analyze the debris field of the lysed cells utilizes biochemical reagents selected from the group consisting of fluorescent ion indicators, luminescent ion indicators, chromogenic enzyme substrates, fluorescent enzyme substrates, luminescent enzyme substrates, fluorescent antibodies, enzyme labeled antibodies, luminescent antibodies, molecular beacons, genetic analysis devices, fluorescent nucleic acids, and luminescent nucleic acids. 19. The device of claim 14, in which the biochemistry analyzer corrects for the distorting effects caused by the variable volume of the cell lysis field by also monitoring the fluorescence or luminescence distribution of a cell cytoplasm tracking dye that is released upon cell lysis. 20. The device of claim 14, further containing a processor connected to the transit analyzer and the cell lyser; in which the processor use data obtained from the transit analyzer to control the operation of the cell lyser. 21. The device of claim 14, further containing a processor connected to the transit analyzer; wherein the processor obtains a first set of data pertaining to the cell surface markers or the cell morphological characteristics of the individual cells;the processor also being connected to the biochemistry analyzer to biochemically analyze the cytoplasm or nuclear debris field of the lysed cells;wherein the processor obtains a second set of data pertaining to the one or more molecules present in the cell cytoplasm or the nucleus of the individual cells;in which the processor correlates the first set of data and the second set of data according to user determined criteria and store or transmit the correlated data. 22. A device for correlating an individual cell's cell surface markers or cell morphological characteristics with one or more molecules present in the cell cytoplasm or nucleus of the individual cells; the device being capable of operating on a plurality of individual cells selected from a population of cells;the device comprising;one or more microfluidic pathways;the microfluidic pathways containing a moving fluid stream capable of transporting the individual cells through at least a portion of the microfludic pathways in an intact state;a transit analyzer to analyze the cell surface markers or the cell morphological characteristics of the individual cells while the cells are transiting the microfluidic pathways in an intact state;a cell lyser to lyse the individual cells while the cells are transiting the microfluidic pathways;a biochemistry analyzer to biochemically analyze the cytoplasmic or nuclear debris field of the lysed cells for the one or more molecules present in the cell cytoplasm or the nucleus of the individual cell; anda processor configured to process data collected from the transit analyzer to analyze the cell surface markers or the cell morphological characteristics to operate either the cell lyser or the cell biochemistry analyzer. 23. The device of claim 22, wherein the processor is configured to obtain a first set of data pertaining to the cell surface markers or the cell morphological characteristics of the individual cells; wherein the processor is configured to obtain a second set of data pertaining to the one or more molecules present in the cell cytoplasm or the nucleus of the individual cells;in which the processor is configured to correlate the first set of data and the second set of data according to user determined criteria and store or transmit the correlated data. 24. The device of claim 22, wherein the processor is configured to obtain a first set of data pertaining to the cell surface markers or the cell morphological characteristics of the individual cells, compare the first set of data with one or more preset user criteria, and vary the operation of the cell lyser to lyse the individual cells; wherein cells that meet one or more preset user criteria are lysed, and wherein cells that fail to meet one or more preset user criteria are not lysed. 25. The device of claim 22, wherein the processor is configured to obtain a first set of data pertaining to the cell surface markers or the cell morphological characteristics of the individual cells, or wherein the processor is configured to obtain a second set of data pertaining to the one or more molecules present in the cell cytoplasm or nucleus of the individual cells; and wherein processor is configured to compare said first data or said second data with one or more preset user criteria, and alter the flow speed or flow direction of said cells or said cell debris through said microfluidic pathways when said first set of data or said second set of data meet one or more of said preset user criteria.