	US 11,939,626 B2
	Methods and systems for performing digital assays using polydisperse droplets
Daniel T. Chiu, Seattle, WA (US); Jason E. Kreutz, Seattle, WA (US); Gloria S. Yen, Seattle, WA (US); and Bryant S. Fujimoto, Seattle, WA (US)
Assigned to UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION, Seattle, WA (US)
Filed by UNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION, Seattle, WA (US)
Filed on Jul. 19, 2022, as Appl. No. 17/813,415.
Application 17/813,415 is a continuation of application No. 16/812,173, filed on Mar. 6, 2020, granted, now 11,427,857.
Application 16/812,173 is a continuation of application No. 15/301,798, granted, now 10,619,192, previously published as PCT/US2015/024840, filed on Apr. 8, 2015.
Claims priority of provisional application 62/047,570, filed on Sep. 8, 2014.
Claims priority of provisional application 61/976,918, filed on Apr. 8, 2014.
Prior Publication US 2022/0356512 A1, Nov. 10, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6816 (2018.01); G01N 15/14 (2006.01); G01N 15/1434 (2024.01); G01N 21/47 (2006.01); G01N 21/64 (2006.01); G06T 7/62 (2017.01); G06V 20/69 (2022.01)

CPC C12Q 1/6816 (2013.01) [G01N 15/1434 (2013.01); G01N 21/47 (2013.01); G01N 21/6428 (2013.01); G06T 7/62 (2017.01); G06V 20/695 (2022.01); G01N 2015/1445 (2013.01); G01N 2021/6439 (2013.01); G06T 2207/10056 (2013.01); G06T 2207/30072 (2013.01)]

19 Claims

1. A method for performing a digital assay, the method comprising:

producing a plurality of polydisperse droplets, wherein at least some of droplets of the plurality of polydisperse droplets comprise a sample, the sample comprising a target molecule;

amplifying the sample to produce an amplified product, wherein amplifying the sample comprises amplifying the target molecule or molecules associated with the target molecule;

associating the amplified product with a detectable agent;

obtaining an image stack of the plurality of polydisperse droplets, wherein the image stack comprises a plurality of images from a plurality of focal planes;

measuring a volume of a droplet of the plurality of polydisperse droplets using the plurality of the images;

detecting from the image stack a presence or absence of the amplified product and the detectable agent in the droplet; and

determining the concentration of the target molecule in the plurality of polydisperse 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 plurality of polydisperse droplets.