Digital assay

Patent metadata

Field	Value
Publication number	US-12569851-B2
Application number	US-201816484620-A
Country	US
Kind code	B2
Filing date	Feb 8, 2018
Priority date	Feb 8, 2017
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

Abstract

Provided herein is a method and device for partitioning a fluidic sample. The device contains a plate containing microwells. The method comprises depositing a sample on one or both of the plates when the plates are in an open configuration, wherein the deposition is in the form of a single or multiple droplet of the sample, wherein at least one of the droplets has a volume that occupies more than two microwells and closing the plates to the closed configuration to partition the sample in the microwells.

First claim

The invention claimed is:

1 . A device for performing a digital assay of a fluidic sample containing or suspected of containing an analyte, comprising:
a first plate, a second plate, spacers, and microwells, wherein: (a) the first and second plates are movable relative to each other into an open configuration and a closed configuration, and comprise, on their respective inner surface, a sample contact area for contacting a fluidic sample that has a volume; (b) the second plate comprises, in the sample contact area, the microwells, wherein each microwell has (i) a predetermined and known geometry, (ii) a well depth of 200 μm or less, and (iii) a volume substantially less than the volume of the fluidic sample; and (c) the spacers are on one or both of the plates, wherein each spacer has a pillar shape and uniform height; wherein in the open configuration, an average spacing between an inner surface of the first plate and a rim of the microwells in the second plate is larger than the well depth, and the sample is deposited on one or both of the plates; wherein in the closed configuration, the inner surface of the first plate faces the inner surface of the second plate, at least a part of the sample is inside the microwells, and the height of the spacers is selected to make the average spacing between the inner surface of the first plate and the rim of the microwells in the second plate to be ½ (one half) of the microwell depth or less; and wherein the device further comprises second spacers that are configured to regulate the spacing between the first and second plates.

2 . An apparatus comprising a thermal cycler and the device of claim 1 .

3 . An apparatus comprising a thermal cycler, the device of claim 1 , and a reader for real-time PCR.

4 . The device of claim 1 , further comprising a binding site that is located on the sample contact area of one or both of the plates, wherein the binding site comprises a capture agent immobilized at the site, and the capture agent is configured to specifically capture the analyte in the sample.

5 . The device of claim 1 , further comprising a surface amplification layer that is located either on the sample contact area of one or both of the plates, wherein the surface amplification layer comprises a capture agent immobilized at the site, and the capture agent is configured to specifically capture the analyte in the sample, wherein the surface amplification layer amplifies an optical signal from the analyte or a label attached to the analyte when the analyte is in proximity of the surface amplification layer rather than a micron or more away.

6 . The device of claim 5 , wherein an amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label that is bound directly or indirectly to the capture agent visible.

7 . The device of claim 1 , wherein device further comprise a reagent in the microwells in a close configuration of the second plate, wherein the reagent generates, when a detection agent specifically binds to the analyte, multiple light emitting components in the microwells.

8 . The device of claim 1 , wherein the average spacing in the closed configuration is configured to make saturation binding time of the analyte to the capture agents 300 sec or less.

9 . The device of claim 1 , wherein the average spacing in the closed configuration is configured to make saturation binding time of the analyte to the capture agents 60 sec or less.

10 . The device of claim 5 , wherein an amplification factor of the surface amplification layer is adjusted to make the optical signal from a single label visible.

11 . The device of claim 4 , wherein the capture agent is a nucleic acid.

12 . The device of claim 4 , wherein the capture agent is a protein.

13 . The device of claim 4 , wherein the capture agent is an antibody.

14 . The device of claim 4 , wherein the capture agent is an aptamer.

15 . The device of claim 5 , wherein the capture agent is an aptamer.

16 . The device of claim 1 , further comprising a storage site that is on the inner surface of one or both of the plates, wherein the storage site comprises a reagent dissolvable in a liquid.

17 . The device of claim 7 , wherein the reagent is a reagent for amplification of an analyte in the sample.

18 . The device of claim 7 , wherein the reagent amplifies the analyte by polymerase chain reaction (PCR).

19 . The device of claim 7 , wherein the reagent is a detection reagent.

20 . The device of claim 1 , wherein each microwell has a volume, and the volume of each microwell is configured, for an expected analyte concentration, so that the distribution of the analyte in each microwell that is filled with the sample follows Poisson distribution.

21 . The device of claim 1 , wherein each microwell has a volume, and the volume of each microwell is configured, for an expected analyte concentration, so that the distribution of analyte in each microwell that is filled with the sample is, on average, one analyte per every 2 microwells, 3 microwells, 5 microwells, 10 microwells, 20 microwells, 30 microwells, 50 microwells, 75 microwells, 100 microwells, 150 microwells, 200 microwells, 300 microwells, 500 microwells, 1000 microwells, 2000 microwells, 10000 microwells, or 100,000 microwells, or in a range of any two value.

22 . The device of claim 1 , wherein each microwell has a volume, and the volume of each microwell is configured for an expected analyte concentration, so that the distribution of analyte in each microwell that is filled with the sample is, on average, one analyte per every 10 microwells, 20 microwells, 30 microwells, 50 microwells, 75 microwells, or 100 microwells, or in a range of any two value.

23 . The device of claim 1 , wherein, in the closed configuration, the average spacing between the inner surface of the first plate and the rim of the microwells in the second plate is less than ½ (one half), ⅓, ⅕, ⅙, 1/7, 18, 1/9, 1/10, 1/11 (one eleventh), 1/20, 1/30, 1/40, 1/50, 1/100, 1/300, 1/500 of the microwell depth.

24 . The device of claim 1 , wherein, in the closed configuration, the inner surface of the first plate and the rim of the microwell in the second plate are in contact.

25 . The device of claim 1 , wherein, in the closed configuration, the average distance between two neighboring microwells is less than 5 nm, 10 nm, 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, or 100 μm.

26 . The device of claim 1 , wherein the microwells have a shape selected from the group consisting of round, rectangle and hexagon, with lattice of square and/or hexagon.

27 . The device of claim 1 , wherein the microwells on the first plate have a period (average well to well center distance) of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, or 1 mm.

28 . The device of claim 1 , wherein the microwells on the second plate have a well size (average length or diameter) of 1 nm to 1 mm.

29 . The device of claim 1 , wherein the microwells on the second plate have a depth of at least 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 50 μm, 500 μm, or 1 mm.

30 . The device of claim 1 , wherein the microwells comprise (i) no metal coating (ii) metal coating on bottom thereof (iii) metal coating on side wall of th

Assignees

• Essenlix Corp

Inventors

• Chou Stephen Y
• Ding Wei
• Zhang Yufan

Classifications

• B01L2300/0819

  Microarrays; Biochips · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• B01L2300/12

  Specific details about materials · CPC title

• B01L2300/0893

  having a very large number of wells, microfabricated wells · CPC title