Method for assaying a micro-object within a microfluidic device
The present disclosure relates to methods for assaying and controlling micro-objects in a microfluidic device. In situ-generated hydrogel barriers are provided for dividing a microfluidic chamber into areas where assaying a cell may be performed without interference from the presence of the cell itself.
1 . A method for preparing a chamber of a microfluidic device to detect an analyte produced within the chamber, the microfluidic device having a microfluidic circuit comprising a flow region and the chamber, wherein the chamber comprises an opening to the flow region, the method comprising:
forming an in situ-generated barrier within the chamber, wherein the in situ-generated barrier defines a first area of the chamber proximal to the opening of the chamber to the flow region and a second area of the chamber that is separated from the first area by the in situ-generated barrier, wherein a portion of the in situ-generated barrier has a thickness that is smaller than a height of the chamber;
disposing a micro-object into the first area of the chamber;
allowing the micro-object to produce the analyte within the first area of the chamber, wherein at least some of the analyte passes through the in situ-generated barrier into the second area of the chamber, wherein the in situ-generated barrier prevents the micro-object from crossing through the situ-generated barrier into the second area;
defining an area of interest within the first area of the second area; and
detecting a signal associated with the analyte within the area of interest.
2 . The method of claim 1 , wherein the in situ-generated barrier comprises one or more discrete sections, each of which is moveably connected to one or more surfaces of the chamber, wherein application of a threshold pressure to the one or more discrete sections of the in situ-generated barrier moves at least one of the one or more discrete sections with respect to the one or more surfaces of the chamber and thereby creates or expands an opening in the second area.
3 . The method of claim 2 , wherein the in situ-generated barrier comprises two or more discrete sections, wherein adjacent sections are separated from one another by a gap.
4 . The method of claim 2 , wherein the in situ-generated barrier consists of two discrete sections which are separated from one another by a gap.
5 . The method of claim 1 , wherein the in situ-generated barrier comprises a non-uniform thickness with respect to an axis of the chamber such that a portion of the in situ-generated barrier is less thick than other portions of the in situ-generated barrier.
6 . The method of claim 5 , wherein the less thick portion of the in situ-generated barrier has a thickness in a z-direction that is smaller than the height of the chamber.
7 . The method of claim 1 , wherein the in situ-generated barrier has a porosity prevents the micro-object from passing through the in situ-generated barrier.
8 . The method of claim 1 , wherein allowing the micro-object to produce the analyte comprises inducing the micro-object to produce the analyte.
9 . The method of claim 8 , wherein inducing the micro-object to produce the analyte comprises introducing a second fluidic medium oxygenated with at least about 10% of oxygen.
10 . The method of claim 1 , further comprising introducing a reporter molecule and allowing the reporter molecule to bind or react with the analyte.
11 . The method of claim 10 , wherein the reporter molecule comprises a first detectable label and detecting the signal comprises detecting a signal associated with the first detectable label.
12 . The method of claim 10 , wherein the in situ-generated barrier has a first permeability with respect to the analyte and a second permeability with respect to the reporter molecule, and wherein the first permeability is lower than the second permeability.
13 . The method of claim 10 , wherein allowing the reporter molecule to bind with the analyte comprises forming a reporter molecule: produced analyte (RMPA) complex, wherein the in situ-generated barrier has a porosity that impedes the RMPA complex from passing through the in situ-generated barrier.
14 . The method of claim 10 , wherein the reporter molecule further comprises a binding component configured to bind the analyte; wherein the binding component of the reporter molecule comprises an amino acid, a polypeptide, a nucleotide, a nucleic acid, or a combination thereof.
15 . The method of claim 11 , further comprising:
introducing a reference molecule into the flow region, wherein the reference molecule comprises a second detectable label different from the first detectable label, and further wherein the reference molecule does not bind the analyte;
allowing the reference molecule to diffuse into the chamber; and
detecting a reference signal associated with the second detectable label.
16 . The method of claim 1 , further comprising exporting the micro-object from the chamber and, optionally, from the microfluidic device.
17 . The method of claim 1 , wherein the in situ-generated barrier comprises a solidified polymer network; wherein the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biological polymer.
18 . The method of claim 1 , further comprising:
introducing a fluorescent dye into the chamber and allowing the fluorescent dye to diffuse within the area of interest; and
detecting a fluorescence signal from the fluorescent dye in the area of interest.
19 . The method of claim 18 , wherein the fluorescent dye is pH sensitive, wherein the fluorescence signal is associated with a pH of a medium within the area of interest.
20 . A method of using a microfluidic device, the microfluidic device having a microfluidic circuit comprising a flow region and a chamber, wherein the chamber comprises an opening to the flow region, the method comprising:
disposing micro-objects into the chamber;
forming an in situ-generated barrier within the chamber, wherein the in situ-generated barrier defines a first area of the chamber proximal to the opening of the chamber to the flow region and a second area of the chamber that is separated from the first area by the in situ-generated barrier, wherein the micro-objects are disposed within the first area of the chamber, and the second area of the chamber is micro-object-free;
allowing the micro-objects to produce an analyte within the first area of the chamber, wherein at least some of the analyte passes through the in situ-generated barrier into the second area of the chamber, wherein the in situ-generated barrier prevents the micro-objects from crossing through the situ-generated barrier into the second area of the chamber;
detecting a signal associated with the analyte within the second area of the chamber; and
exporting the micro-objects out of the chamber.
21 . A method for preparing a chamber of a microfluidic device to detect an analyte produced within the chamber, the microfluidic device having a microfluidic circuit comprising a flow region and the chamber, wherein the chamber comprises an opening to the flow region, the method comprising:
disposing a micro-object into the chamber of the microfluidic device;
forming an in situ-generated barrier within the chamber, wherein the in situ-generated barrier defines a first area of the chamber proximal to the opening of the chamber to the flow region and a second area of the chamber that is separated from the first area by the in situ-generated barrier, wherein the micro-object is disposed within the first area of the chamber, and the second area of the chamber is micro-object-free;
allowing the micro-object to produce the analyte within the first area of the chamber, wherein at least some of the analyte diffuses through the in situ-generated barrier into the second area of the chamber, wherein the in situ-generated barrier prevents the micro-object from crossing through the situ-generated barrier into the second area;
defining an area of interest within the first area or the second area; and
detecting a signal associated with the analyte within the area of interest.
22 . The method of claim 21 , further comprising moving the micro-object into the first area of the chamber prior to forming the in situ-generated barrier.