Droplet-Based Particle Sorting

• US 20150330943A1
• Filed: 07/22/2015
• Published: 11/19/2015
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pollack, Michael G., Pamula, Vamsee K., Srinivasan, Vijay
• Granted Patent: US 9,395,329 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1031 by measuring electrical or magnetic effects
• CPC Class Codes: G01N 2015/019 Biological contaminants; Fouling
• CPC Class Codes: G01N 2015/1028 Sorting particles
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 35/10 Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices G01N35/0099 takes precedence
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/118339 : with formation of a segmented stream
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention relates to droplet-based particle sorting. According to one embodiment, a method of providing a droplet comprising a single cell is provided, wherein the method includes providing a droplet comprising a suspension of cells on an electrowetting droplet actuator surrounded by an oil medium, dispensing from the droplet to provide a dispensed droplet, determining whether the dispensed droplet on the electrowetting droplet actuator comprises a single cell, and sorting the dispensed droplet on the electrowetting droplet actuator based on the results of the determining step using electrowetting-mediated droplet operations.

Claims

1-49. -49. (canceled)

50. A method of providing a droplet comprising a single cell, the method comprising:

(a) providing a droplet comprising a suspension of cells on an electrowetting droplet actuator surrounded by an oil medium;

(b) dispensing from the droplet of (a) to provide a dispensed droplet, wherein the dispensing is electrowetting-mediated;

(c) determining whether the dispensed droplet on the electrowetting droplet actuator comprises a single cell; and

(d) sorting the dispensed droplet on the electrowetting droplet actuator based on the results of the determining step using electrowetting-mediated droplet operations.

51. The method of claim 50 further comprising repeating steps 50(b), 50(c), and 50(d).

52. The method of claim 50 wherein the oil medium is substantially immiscible with the droplets of 50(a) and (b).

53. The method of claim 50 wherein step 50(c) is achieved by detecting a number of cells in the dispensed droplet based on a property of and/or signal from the dispensed droplet.

54. The method of claim 53 wherein the detecting step is achieved using a sensor.

55. The method of claim 50, further wherein a single cell determined to be comprised in the dispensed droplet comprises a target cell type, and wherein step 50(c) further comprises detecting the target cell type of the single cell in the dispensed droplet based on a property of and/or signal detected from the dispensed droplet.

56. The method of claim 50 further comprising combining a dispensed droplet determined to comprise a single cell with a separate droplet provided on the droplet actuator to form a combined droplet, wherein the separate droplet comprises a substance which interacts with the single cell.

57. The method of claim 56 wherein the substance which may interact with the single cell is selected from the group consisting of:

beads, cells, microparticles, nanoparticles, drugs, proteins, peptides, antigens, toxins, and antibodies.

58. The method of claim 56 further comprising determining in the combined droplet the presence or quantity of the substance indicative of the single cell'"'"'s response to the substance.

59. The method of claim 58 wherein determining in the combined droplet the presence or quantity of the substance indicative of the single cell'"'"'s response to the substance comprises conducting an assay comprising electrowetting-mediated droplet operations.

60. The method of claim 56 wherein the single cell comprises a T cell.

61. The method of claim 50 further comprising determining if the dispensed droplet comprises multiple cells and combining a dispensed droplet determined to comprise multiple cells with the droplet comprising the suspension of cells.

62. The method of claim 50 further comprising:

(a) determining if the dispensed droplet comprises multiple cells;

(b) splitting a dispensed droplet determined to comprise multiple cells to yield two or more daughter droplets; and

(c) analyzing one or more of the daughter droplets for the presence of a single cell.

63. The method of claim 50 further comprising:

(a) determining if the dispensed droplet comprises multiple cells;

(b) combining a dispensed droplet determined to comprise multiple cells with a buffer droplet to form a combined droplet;

(c) splitting the combined droplet to yield two or more daughter droplets; and

(d) analyzing one or more of the daughter droplets for the presence of a single cell.

64. The method of claim 50 further comprising determining if the dispensed droplet does not contain a single cell and transporting a dispensed droplet determined not to contain a single cell back to the droplet comprising the suspension of cells using electrowetting-mediated droplet operations.

65. The method of claim 50 further comprising determining if the dispensed droplet does not contain a single cell and discarding a dispensed droplet determined not to contain a single cell.

66. The method of claim 50 wherein step 50(c) comprises detecting if a signal indicative of a presence of a single cell is present in the dispensed droplet.

67. The method of claim 66 wherein the signal from a dispensed droplet determined to comprise a single cell comprises a fluorescence from a molecule which binds to the single cell.

68. The method of claim 66 wherein the signal from a dispensed droplet determined to comprise a single cell comprises a radioactive signal.

69. The method of claim 66 wherein the signal from a dispensed droplet determined to comprise a single cell comprises a visual image.

70. The method of claim 50 wherein a dispensed droplet determined to comprise a single cell is transported to an on-droplet microactuator reservoir.

71. The method of claim 50 wherein additional droplet operations are conducted on a dispensed droplet determined to comprise a single cell.

72. The method of claim 50 wherein a dispensed droplet determined to comprise a single cell is further analyzed.