Biochemical analysis of partitioned cells
First Claim
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1. A device that generates single cell aqueous reaction volumes in a flow channel, the device comprising:
- a) the flow channel;
b) a first source of an aqueous solution that comprises a plurality of cells, which source is fluidly coupled to the flow channel;
c) a second source of a partitioning solution that is immiscible in the aqueous solution, which second source is fluidly coupled to the flow channel;
d) a controller operably coupled to the first and second sources, which controller directs flow of the aqueous solution from the first source into the flow channel, and the partitioning solution from the second source into the flow channel;
whereby the partitioning solution partitions cells in the aqueous solution into separate aqueous reaction volumes;
e) an excitation light source capable of illuminating the aqueous reaction volumes in said flow channel at a desired wavelength;
f) a spectrophotometric detector capable of detecting a light emission signal from a single aqueous reaction volume in said flow channel; and
,g) an electronic module operably connected to the detector for collecting and storing said signals detected by the detector from a plurality of aqueous reaction volumes.
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Abstract
The invention relates to compositions and methods for the analysis of biomolecules associated with cells, where the presence or absence of a particular biomolecule (e.g., an expressed protein or a nucleic acid gene expression product) associated with the cells is examined. The invention provides methods for single cell biochemical analysis, as well as instrumentation for the single-cell biochemical analysis. Most advantageously, the invention affords methods and instrumentation for high-throughput biochemical analysis of large numbers of single cells.
202 Citations
79 Claims
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1. A device that generates single cell aqueous reaction volumes in a flow channel, the device comprising:
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a) the flow channel; b) a first source of an aqueous solution that comprises a plurality of cells, which source is fluidly coupled to the flow channel; c) a second source of a partitioning solution that is immiscible in the aqueous solution, which second source is fluidly coupled to the flow channel; d) a controller operably coupled to the first and second sources, which controller directs flow of the aqueous solution from the first source into the flow channel, and the partitioning solution from the second source into the flow channel;
whereby the partitioning solution partitions cells in the aqueous solution into separate aqueous reaction volumes;e) an excitation light source capable of illuminating the aqueous reaction volumes in said flow channel at a desired wavelength; f) a spectrophotometric detector capable of detecting a light emission signal from a single aqueous reaction volume in said flow channel; and
,g) an electronic module operably connected to the detector for collecting and storing said signals detected by the detector from a plurality of aqueous reaction volumes. - View Dependent Claims (2, 3, 4)
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5. A method for detecting the presence or absence of a biomolecule associated with a plurality of cells, the method comprising:
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a) providing; i) said plurality of cells in an aqueous first solution, said first solution further comprising at least one reagent for detecting the presence or absence of the biomolecule, wherein the reagent is capable of generating or participating in generating an amplified signal corresponding to the presence or absence of the biomolecule; ii) a second solution that is immiscible with said first solution and capable of forming vesicles when admixed with the first solution; b) combining the first solution and the second solution under conditions that result in the formation of a plurality of reaction vesicles, wherein said reaction vesicles thus formed each comprise, (i) one cell from the plurality of cells, and (ii) said reagent for detecting the presence or absence of the biomolecule; c) subjecting said reaction vesicles to conditions suitable for detecting the biomolecule, wherein the reagent generates or participates in generating an amplified signal corresponding to the presence or absence of the biomolecule associated with the one cell in each reaction vesicle; d) detecting said amplified signal from the plurality of vesicles; e) correlating the signal with the presence or absence of the biomolecule associated with said cells, thereby detecting the presence or absence of the biomolecule associated with said cells and generating a detection result; and f) displaying the detection result for said plurality of cells. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for detecting the presence or absence of a biomolecule associated with a plurality of cells, the method comprising:
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a) providing; i) said plurality of cells in an aqueous first solution, said first solution further comprising at least one reagent for detecting the presence or absence of the biomolecule, wherein the reagent is capable of generating or participating in generating an amplified signal corresponding to the presence or absence of the biomolecule; ii) a second solution that is immiscible with said first solution; b) channeling said aqueous solution through a liquid flow system, thereby generating a channeled liquid flow, said liquid flow system further comprising a means for delivering said second solution into the channeled liquid flow at intervals, thereby generating a plurality of partitioned aqueous reaction volumes in the channeled liquid flow, said partitioned aqueous reaction volumes separated from each other by partitions comprising the injected second solution, wherein each partitioned aqueous reaction volume thus formed comprises (i) said at least one cell from the plurality of cells, and (ii) said reagent for detecting the presence or absence of the biomolecule; c) subjecting the partitioned aqueous reaction volumes to conditions suitable for detecting the biomolecule, wherein the reagent generates or participates in generating an amplified signal corresponding to the presence or absence of the biomolecule associated with the one cell in each partitioned aqueous reaction volume, thereby generating a signal corresponding to the presence or absence of the biomolecule in the partitioned aqueous reaction volume; d) detecting said amplified signal form the plurality of partitioned aqueous reaction volumes; e) correlating the signal with the presence or absence of the biomolecule associated with said cells, thereby detecting the presence or absence of the biomolecule associated with said cells and generating a detection result; and f) displaying the detection result for said plurality of cells. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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61. A composition comprising:
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a) a plurality of aqueous reaction core solutions, each comprising; (i) a cell, and (ii) at least one reagent for detecting the presence or absence of a biomolecule associated with said cell, wherein the reagent is capable of generating or participating in generating an amplified signal corresponding to the presence or absence of the biomolecule; and b) an immiscible liquid shell that partitions the aqueous reaction core solution. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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Specification