Methods and Devices for Correlated, Multi-Parameter Single Cell Measurements and Recovery of Remnant Biological Material
First Claim
1. An integrated structure for microfluidic single-cell analysis and correlating comprising:
- a cartridge, the cartridge comprisingan optical window,a plurality of reservoirs, including at least;
a sample reservoir,a fluid reservoir, anda reagent reservoir; and
a chip, the chip including at least;
a cell inlet channel, the cell inlet channel being fluidically coupled to the sample reservoir,a first fluid inlet channel fluidically coupled to the fluid reservoir and the cell inlet channel,a second fluid inlet channel fluidically coupled to the reagent reservoir and the cell inlet channel,a serpentine channel comprising a first end which is fluidically coupled to the cell input channel downstream of the first and second fluid inlet channels and a plurality of parallel partitions having first and second ends and being fluidically connected to each other,a plurality of venting vias located at the first and second ends of the plurality of partitionsthe chip being disposed adjacent the optical window.
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Abstract
Methods and apparatus are provided for analysis and correlation of phenotypic and genotypic information for a high throughput sample on a cell by cell basis. Cells are isolated and sequentially analyzed for phenotypic information and genotypic information which is then correlated. Methods for correlating the phenotype-genotype information of a sample population can be performed on a continuous flow sample within a microfluidic channel network or alternatively on a sample preloaded into a nano-well array chip. The methods for performing the phenotype-genotype analysis and correlation are scalable for samples numbering in the hundreds of cells to thousands of cells up to the tens and hundreds of thousand cells.
164 Citations
84 Claims
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1. An integrated structure for microfluidic single-cell analysis and correlating comprising:
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a cartridge, the cartridge comprising an optical window, a plurality of reservoirs, including at least; a sample reservoir, a fluid reservoir, and a reagent reservoir; and a chip, the chip including at least; a cell inlet channel, the cell inlet channel being fluidically coupled to the sample reservoir, a first fluid inlet channel fluidically coupled to the fluid reservoir and the cell inlet channel, a second fluid inlet channel fluidically coupled to the reagent reservoir and the cell inlet channel, a serpentine channel comprising a first end which is fluidically coupled to the cell input channel downstream of the first and second fluid inlet channels and a plurality of parallel partitions having first and second ends and being fluidically connected to each other, a plurality of venting vias located at the first and second ends of the plurality of partitions the chip being disposed adjacent the optical window. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. An integrated structure for microfluidic single-cell analysis and correlating comprising:
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a cartridge, the cartridge comprising an optical window, a plurality of reservoirs, including at least; a sample reservoir, a fluid reservoir, and a reagent reservoir; a lid, the lid including at least; at least one pneumatic pressure port, the port having an inlet and being coupled to at least one of the sample reservoir and the fluid reservoir, and a filter disposed between the inlet of the pneumatic pressure port and at least one of the sample reservoir and the fluid reservoir, and a manifold, the manifold coupling pneumatic pressure from a source to the pneumatic pressure port without intervening tubing. a chip, the chip including at least; a cell inlet channel adapted to receive one or more cells in a fluidic medium, the cell inlet channel being fluidically coupled to the sample reservoir, a first fluid inlet channel fluidically coupled to the fluid reservoir and the cell inlet channel, a second fluid inlet channel fluidically coupled to the reagent reservoir and the cell inlet channel, an outlet channel fluidically coupled to the cell inlet channel downstream of the first fluid inlet channel and the second fluid inlet channel the chip being at least partially disposed adjacent the optical window; and a capillary tube fluidically coupled to the outlet channel. - View Dependent Claims (26, 27, 28)
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29. A microfluidic chip for single cell analysis and correlation comprising:
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a loading zone comprising; a cell inlet channel, the cell inlet channel configured to be fluidically coupled to a sample source, a first fluid inlet channel having first and second ends, the first end configured to be fluidically coupled to reagent source and the second end fluidically coupled to the cell inlet channel; and a second fluid inlet channel having first and second ends, the first end configured to be fluidically coupled to fluid source and the second end fluidically coupled to the cell inlet channel downstream of the first fluid inlet channel; an analysis zone comprising a serpentine channel comprising a first end which is fluidically coupled to the cell input channel downstream of the first and second fluid inlet channels and a plurality of parallel partitions having first and second ends and being fluidically connected to each other, a plurality of venting vias located at the first and second ends of the plurality of partitions. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38)
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39. A method of correlating phenotypic and genotypic information on a cell by cell basis comprising:
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providing a first solution containing a plurality of cells and at least one reagent for amplifying a target DNA sequence and a second solution that is immiscible with the first solution; sequentially analyzing the phenotype of each cell in the first solution; combining the first solution and second solution such that a stream comprising a plurality of nanoliter microvessels are formed, wherein a majority of the nanoliter microvessels encapsulate a single cell or remain empty of cells; encoding the stream of nanoliter microvessels with a reference signal; subjecting the nanoliter microvessels to thermal conditions suitable to amplify the target DNA; measuring gene expression in each microreactor; and decoding the reference signal to correlate the measurement of gene expression from each microreactor with the phenotype of the cell in the microreactor. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
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63. An integrated system for providing correlated phenotypic and genotypic analysis on individual cells in a microfluidic network comprising:
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a sample source comprising an aqueous solution containing a plurality of cells; an encapsulation source comprising a hydrophobic fluid; a microfluidic network comprising; a loading zone comprising; a sample flow channel fluidically coupled to the sample source; a lateral flow channel fluidically coupled to the fluid reservoir and the sample flow channel, and a controller configured to direct flow conditions in the lateral flow channel such that the sample source is partitioned into a plurality of nanoliter microvessels, and an analysis zone comprising; a serpentine channel comprising a first end which is fluidically coupled to the cell input channel downstream of the reagent inlet and lateral flow channels and a plurality of parallel partitions having first and second ends and being fluidically connected to each other, a plurality of venting vias located at the first and second ends of the plurality of partitions a first optical detector operably coupled to the sample flow channel, the detector configured to measure a signal from the nanoliter microvessels in the sample flow channel; a thermal module comprising a heating element and a thermal control element operably coupled to the analysis zone; an excitation light source configured to illuminate the serpentine channel at a certain wavelength; a second optical detector configured to detect and measure an amplified product from the nanoliter microvessels in the serpentine channel; and a processor connected to the optical detectors for recording and correlating signals detected by the first and second optical detectors. - View Dependent Claims (64, 65, 66, 67, 68, 69, 70, 71, 72)
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73. A method of correlating phenotypic and genotypic information on a cell by cell basis in a microfluidic environment comprising:
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providing a sample solution containing a plurality of cells in an aqueous environment and an encapsulation solution that is immiscible with the sample solution; introducing the sample solution and the encapsulation solution into a microfluidic network; sequentially measuring the phenotype of each cell in the sample solution; combining the sample solution and the encapsulation solution within the microfluidic network such that such that the sample solution is partitioned into a plurality of nanoliter microvessels; encoding the stream of nanoliter microvessels with a reference signal; loading the plurality of nanoliter microvessels in a serpentine channel comprising a plurality of parallel partitions having first and second ends and being fluidically connected to each other and a plurality of venting vias located at the first and second ends of the plurality of partitions; subjecting the serpentine channel to repetitive temperature cycling to amplify a target DNA sequence in the plurality of nanoliter microvessels; measuring the amplified product in the plurality of nanoliter microvessels; and decoding the reference signal to correlate the measurement of amplified product from each microreactor with the phenotype measurement of the cell in each microreactor. - View Dependent Claims (74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)
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Specification