Methods and apparatus for performing digital assays using polydisperse droplets
Methods, devices, and systems for performing digital assays are provided. In certain aspects, the methods, devices, and systems can be used for the amplification and detection of nucleic acids. In certain aspects, the methods, devices, and systems can be used for the recognition, detection, and sizing of droplets in a volume. Also provided are compositions and kits suitable for use with the methods and devices of the present disclosure.
1. A method for performing a digital assay, the method comprising:
producing a plurality of polydisperse droplets, wherein at least some of the droplets comprise a sample, the sample comprising a target molecule;
amplifying the sample to produce an amplified product;
associating the amplified product with a detectable agent;
obtaining an image stack for a droplet, wherein the droplet is from the plurality of polydisperse droplets, and wherein the image stack comprises a plurality of images from a plurality of focal planes;
measuring the volume of the droplet using a plurality of the images from the image stack;
detecting from the image stack the presence or absence of the detectable agent in the droplet; and
determining the concentration of the target molecule in the plurality of droplets based on the presence or absence of the detectable agent and the amplified product in the plurality of droplets and the volume of at least some of the droplets.
2. The method of claim 1 , further comprising: forming an emulsion comprising an aqueous phase and a non-aqueous phase.
3. The method of claim 1 , wherein the plurality of polydisperse droplets further comprises a fluid interface modification element.
4. The method of claim 3 , wherein the fluid interface modification element is a surfactant.
5. The method of claim 1 , wherein amplifying the sample comprises performing polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA), loop-mediated amplification (LAMP), or any combination thereof.
6. The method of claim 1 , wherein the distribution of droplet diameters has a standard deviation greater than 5% of the mean droplet diameter.
7. The method of claim 1 , wherein the volumes in the polydisperse droplets vary by more than a factor of 2.
8. A method for performing a digital assay, the method comprising:
producing a plurality of polydisperse droplets having a distribution of droplet diameters with a standard deviation greater than 5% of the mean droplet diameter, wherein at least some of the droplets comprise a sample, the sample comprising a target molecule;
amplifying the sample to produce an amplified product;
associating the amplified product with a detectable agent;
imaging the plurality of polydisperse droplets to obtain a plurality of images with a plurality of focal planes for each droplet of the plurality of polydisperse droplets;
measuring the volume of at least one droplet using a plurality of the images from the plurality of focal planes, wherein the at least one droplet is from the plurality of polydisperse droplets;
detecting the presence or absence of the detectable agent in the at least one droplet; and
determining the concentration of the target molecule in the plurality of droplets based on the presence or absence of the detectable agent and the amplified product in the plurality of polydisperse droplets and the volume of at least some of the droplets.
9. The method of claim 1 , wherein the presence or absence of the detectable agent in the droplet is detected from at least one of the plurality of focal planes in the image stack.
10. The method of claim 8 , wherein the at least one droplet comprises at least 1,000 droplets.
11. The method of claim 1 , wherein the measuring the volume of the droplet further comprises correlating between a plurality of images of the image stack and a largest diameter of the droplet and determining the volume of the droplet therefrom.
12. The method of claim 8 , wherein the measuring the volume of the at least one droplet further comprises correlating between a plurality of images of the image stack and a largest diameter of the droplet and determining the volume of the droplet therefrom.
13. The method of claim 1 , wherein the measuring the volume of the droplet further comprises extrapolating a largest diameter of the droplet from a plurality of images of the image stack and determining the volume of the droplet therefrom.
14. The method of claim 8 , wherein the measuring the volume of the at least one droplet further comprises extrapolating a largest diameter of the droplet from a plurality of images of the image stack and determining the volume of the droplet therefrom.
15. The method of claim 1 , wherein the sample comprises a nucleotide.
16. The method of claim 1 , wherein the sample comprises a nucleic acid amplification reagent selected from: a thermostable DNA polymerase, a nucleotide, a primer, a probe, or any combination thereof.
17. The method of claim 1 , wherein the plurality of polydisperse droplets comprises a fluid interface modification element.
18. The method of claim 17 , wherein the fluid interface modification element comprises a surfactant.
19. The method of claim 1 , wherein the method is performed in a multi-well plate.
20. The method of claim 1 , wherein the detecting comprises imaging with an optical imaging source.
21. The method of claim 20 , wherein the optical imaging source is configured to perform one or more of confocal microscopy, line confocal microscopy, deconvolution microscopy, spinning disk microscopy, multi-photon microscopy, planar illumination microscopy, Bessel beam microscopy, differential interference contrast microscopy, phase contrast microscopy, epiflouorescent microscopy, bright field imaging, dark field imaging, oblique illumination, or any combination thereof.
22. The method of claim 1 , wherein the measuring the volume of the droplet further comprises:
identifying at least one pixel set in at least one image of the image stack;
identifying the at least one pixel set as corresponding to at least a part of at least one droplet;
identifying at least one individual droplet based on the correspondence; and
measuring the volume of the identified at least one individual droplet based on the at least one pixel set.