Methods, systems and kits for in-pen assays
Methods, systems and kits are described herein for detecting the results of an assay. In particular, the methods, systems and devices of the present disclosure rely on a difference between the diffusion rates of a reporter molecule and an analyte of interest in order to quantify an amount of analyte in a microfluidic device. The analyte may be a secreted product of a biological micro-object.
1. A method of assessing a level of secretion of an analyte by a biological micro-object, or a population of biological micro-objects generated therefrom, the method comprising:
introducing the biological micro-object into a sequestration pen of a microfluidic device, wherein the microfluidic device comprises an enclosure having a flow region, wherein the sequestration pen is fluidically connected to the flow region, and wherein the sequestration pen contains a first fluidic medium;
allowing the biological micro-object, or the population of biological micro-objects generated therefrom, to secrete an analyte comprising an exogenous tag into the first fluidic medium within the sequestration pen;
introducing a second fluidic medium into the flow region, wherein the second fluidic medium comprises a plurality of reporter molecules, and wherein each reporter molecule comprises:
a binding component configured to bind the exogenous tag of the secreted analyte; and
a detectable label;
allowing a portion of the plurality of reporter molecules to diffuse into the sequestration pen and bind to the analyte secreted therein, thereby producing a plurality of reporter molecule: secreted analyte (RMSA) complexes; and
detecting reporter molecules located within an area of interest within the microfluidic device, wherein the area of interest includes at least a portion of the sequestration pen.
2. The method of claim 1 , wherein the sequestration pen has an isolation region and a connection region fluidically connecting the isolation region to the flow region, and wherein the isolation region and the connection region are configured such that components of a fluidic medium in the isolation region are exchanged with components of a fluidic medium in the flow region substantially only by diffusion.
3. The method of claim 1 , wherein the biological micro-object is a biological cell, and wherein the method further comprises expanding the biological cell within the sequestration pen into a clonal population of biological cells.
4. The method of claim 1 , wherein the detectable label comprises a visible, luminescent, phosphorescent, or fluorescent label.
5. The method of claim 4 , wherein the detectable label of the reporter molecules is a fluorescent label, and wherein said detecting the reporter molecules comprises detecting fluorescence emission from the fluorescent label of the reporter molecules within the area of interest.
6. The method of claim 5 further comprising:
introducing the second fluidic medium into the flow region by flowing the second fluidic medium through the flow region for a first period of time; and
exposing, for a second period of time, a portion of the microfluidic device comprising the sequestration pen to electromagnetic radiation comprising a wavelength which is capable of exciting the fluorescent label of the reporter molecules.
7. The method of claim 6 , wherein:
detecting fluorescence emission within the area of interest is performed after the second period of time;
detecting fluorescence emission within the area of interest is performed two or more times during a third period of time; and/or
detecting fluorescence emission within the area of interest is performed substantially continuously during a third period of time.
8. The method of claim 7 further comprising:
exposing, for a fourth period of time, a portion of the microfluidic device comprising at least a portion of the sequestration pen to electromagnetic radiation but not the flow region, wherein the fourth period of time is sufficient to photobleach the fluorescent label of any reporter molecules present in the portion of the sequestration pen; and
detecting fluorescence emission within the photobleached portion of the sequestration pen.
9. The method of claim 8 , wherein:
detecting fluorescence emission within the photobleached portion of the sequestration pen is performed after the fourth period of time;
detecting fluorescence emission within the photobleached portion of the sequestration detecting fluorescence emission within the photobleached portion of the sequestration pen is performed two or more times during a fifth period of time; and/or
detecting fluorescence emission within the photobleached portion of the sequestration detecting fluorescence emission within the photobleached portion of the sequestration pen is performed substantially continuously during a fifth period of time.
10. The method of claim 8 , wherein said steps of exposing for a fourth period of time and detecting fluorescence emission within the photobleached portion of the sequestration pen are repeated one or more times.
11. The method of claim 1 , further comprising:
introducing a third fluidic medium into the flow region, wherein the third fluidic medium does not comprise any of the reporter molecules; and
allowing at least a portion of unbound reporter molecules to diffuse out of the sequestration pen,
wherein detecting the reporter molecules located within the area of interest occurs at a time selected such that an amount of unbound reporter molecules that have diffused out of the sequestration pen is at least 2× greater than an amount of RMSA complexes that have diffused out of the sequestration pen.
12. The method of claim 1 , further comprising quantifying the level of secretion of the analyte and/or providing a secretion score for the sequestration pen.
13. The method of claim 1 , wherein the exogenous tag of the secreted analyte comprises a peptide sequence.
14. The method of claim 13 , wherein the peptide sequence comprises a FLAG epitope, a polyhistidine sequence, a hemagglutinin (HA) epitope, or a Myc epitope.
15. The method of claim 13 , wherein the peptide sequence comprises the amino acid sequence (from N-terminal to C-terminal) DYKDDDDK (SEQ ID NO: 11).
16. The method of claim 13 , wherein the peptide sequence comprises the amino acid sequence (from N-terminal to C-terminal) HHHHHH (SEQ ID NO: 12).
17. The method of claim 13 , wherein the binding component of the reporter molecule is an antibody.
18. The method of claim 16 , wherein the binding component of the reporter molecule comprises a chelating agent.
19. The method of claim 1 , wherein the analyte secreted by the biological micro-object comprises a protein.
20. The method of claim 19 , wherein the analyte secreted by the biological micro-object is an antibody.
21. The method of claim 1 , wherein the microfluidic device comprises a plurality of sequestration pens, wherein a biological micro-object is introduced into at least two sequestration pens of the plurality, and wherein the remainder of the method is carried out with respect to each of the at least two sequestration pens.
22. The method of claim 21 , further comprising comparing a level of secretion for sequestration pens of the at least two sequestration pens of the plurality.
23. The method of claim 21 , further comprising:
selecting one or more of the at least two sequestration pens; and
exporting one or more biological micro-objects from each of the selected sequestration pens out of the microfluidic device.