Microfluidic devices having isolation pens and methods of testing biological micro-objects with same

Abstract

A microfluidic device can comprise at least one swept region that is fluidically connected to unswept regions. The fluidic connections between the swept region and the unswept regions can enable diffusion but substantially no flow of media between the swept region and the unswept regions. The capability of biological micro-objects to produce an analyte of interest can be assayed in such a microfluidic device. Biological micro-objects in sample material loaded into a microfluidic device can be selected for particular characteristics and disposed into unswept regions. The sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Flows of medium in the swept region do not substantially affect the biological micro-objects in the unswept regions, but any analyte of interest produced by a biological micro-object can diffuse from an unswept region into the swept region, where the analyte can react with the assay material to produce a localized detectable reaction. Any such detected reactions can be analyzed to determine which, if any, of the biological micro-objects are producers of the analyte of interest.

1 - 28 . (canceled) 
     
     
         29 . A microfluidic device comprising:
 a flow region configured to contain a flow of a first fluidic medium; and   a microfluidic sequestration pen having:
 (a) an isolation region configured to contain a second fluidic medium, and 
 (b) an opening into said flow region, wherein said sequestration pen is configured to permit mixing of components of said second medium with said first medium by diffusion or mixing of components of said first medium with said second medium by diffusion, and further wherein said isolation region is a non-flow region of said microfluidic device. 
   
     
     
         30 . The microfluidic device of  claim 29 , wherein said opening is a single opening that opens laterally into said microfluidic channel. 
     
     
         31 . The microfluidic device of  claim 29 , wherein a height H ch  of said microfluidic channel at said single opening is between 20 to 100 micrometers. 
     
     
         32 . The microfluidic device of  claim 29 , wherein said microfluidic device further comprises a first electrode, an electrode activation substrate, and a second electrode, wherein said first electrode is part of a first wall of an enclosure within the microfluidic device and said electrode activation substrate and said second electrode is part of a second wall of said enclosure. 
     
     
         33 . The microfluidic device of  claim 32 , wherein said electrode activation substrate is light-activated. 
     
     
         34 . The microfluidic device of  claim 33 , wherein said electrode activation substrate comprises:
 (i) a photoconductive material, and said electrode activation substrate comprises DEP electrodes, or   (ii) a semiconductor material comprising a plurality of doped layers, electrically insulating layers and electrically conductive layers forming semiconductor integrated circuits, thereby providing DEP electrodes at electrode regions at an inner surface of said electrode activation substrate.   
     
     
         35 . The microfluidic device of  claim 32 , wherein said first wall of said microfluidic device is a cover and said second wall of said microfluidic device is a base. 
     
     
         36 . The microfluidic device of  claim 29 , wherein barriers defining said microfluidic sequestration pen extends from a surface of a base of said microfluidic device to an upper wall of said microfluidic device opposite said surface. 
     
     
         37 . The microfluidic device of  claim 29 , wherein a volume of said isolation region is 1×10 5  cubic microns or more. 
     
     
         38 . The device of  claim 30 , wherein said single opening is perpendicular to a direction of said flow of said first medium in said flow region. 
     
     
         39 . A computer program product comprising one or more non-transitory computer-readable media having computer instructions stored therein, the computer program instructions being configured such that, when executed by one or more computing devices, the computer program instructions cause the one or more computing devices to:
 flow one or more biological cells into a flow channel of a microfluidic device, the flow channel configured to contain a flow of a first fluidic medium;   load the one or more biological cells within the flow channel into an isolation region of a sequestration pen having an opening to the flow region, the isolation region configured to contain a second fluidic medium;   allow for incubation of said one or more biological cells loaded into the isolation region of the sequestration pen, thereby allowing for production of an analyte of interest from said one or more biological cells;   dispose capture micro-objects in said flow channel, at a location adjacent to the opening of said sequestration pen to said flow channel, said capture micro-objects comprising an affinity agent capable of specifically binding said analyte of interest; and   monitor binding of said capture micro-objects to said analyte of interest.   
     
     
         40 . The computer program product of  claim 39 , wherein the computer program instructions for loading the one or more biological cells within the flow channel into an isolation region includes computer program instructions to cause the one or more computing devices to:
 adjust the flow of the first fluidic medium;   capture an image of the flow channel having the one or more biological cells therein after adjusting the flow of the first fluidic medium; and   move the one or more biological cells into the isolation region of the sequestration pen based on the image captured of the flow channel.   
     
     
         41 . The computer program product of  claim 40 , wherein the computer program instructions for moving the one or more biological cells includes computer program instructions to cause the one or more computing devices to project a pattern of light onto an inner surface of the flow region. 
     
     
         42 . The computer program product of  claim 39 , the computer program instructions cause the one or more computing devices to:
 determine a path for the one or more biological cells from the flow channel to the sequestration pen, and wherein loading the one or more biological cells is based on the path.   
     
     
         43 . A system comprising:
 a flow controller configured to adjust flow of a first fluidic medium having a micro-object therein that is provided to a flow channel of a microfluidic device;   an imaging device configured to capture images of the microfluidic device; and   a control circuit configured to instruct the flow controller to adjust the flow of the first fluidic medium, instruct the imaging device to capture an image of the flow channel after the flow of the first fluidic medium is adjusted, and move the micro-object from the flow channel to an isolation region of a sequestration pen having an opening into the flow channel based on the image, thereby permitting mixing of components of said second medium with said first medium by diffusion or mixing of components of said first medium with said second medium by diffusion.   
     
     
         44 . The system of  claim 43 , wherein said isolation region is a non-flow region of said microfluidic device. 
     
     
         45 . The system of  claim 43 , further comprising:
 a light source configured to generating patterns of light, and wherein the control circuit is further configured to instruct the light source to generate a pattern of light to move the micro-object from the flow channel to the isolation region of the sequestration pen.   
     
     
         46 . The system of  claim 45 , wherein the pattern of light is based on the image captured after the flow of the first fluidic medium is adjusted.