Automated detection of assay-positive areas in microfluidic devices

Patent metadata

Field	Value
Publication number	US-2016160259-A1
Application number	US-201514964025-A
Country	US
Kind code	A1
Filing date	Dec 9, 2015
Priority date	Dec 9, 2014
Publication date	Jun 9, 2016
Grant date	—

Abstract

Methods are provided for the automated detection of assay-positive assay areas in a microfluidic device. When assays are performed in a microfluidic device, the configuration of the microfluidic circuit and its constituent circuit elements can determine where the reagents/analytes used in the assay can be located within the microfluidic circuit. Methods are provided for automatic identification of the size and shape of the assay areas based on a number of parameters which may include type of assay involved, shape and dimensions of microfluidic circuit elements, velocity and physical characteristics of the fluidic medium within the microfluidic circuit, physical/chemical properties of the analytes/reagents, and/or the number of cells being assayed.

First claim

1 . An automated method of detecting an assay-positive assay area within a microfluidic device comprising one or more circuit elements, the method comprising:
collecting a set of digital images I i (i=1 to n) of an automatically-identified assay area AA x , wherein the automatically-identified assay area AA x , is identified based, at least in part, on the dimensions of the one or more circuit elements; calculating a rate of change Δ x over the course of all or part of the assay based on the set of digital images I i of the automatically-identified assay area AA x ; comparing the rate of change Δ x to a threshold value Δ°; and determining that the automatically-identified assay region AA x is assay-positive if Δ x is greater than Δ°.

2 . The method of claim 1 , wherein the microfluidic device comprises a channel and the automatically-identified assay area AA x is identified based, at least in part, on the dimensions of the channel.

3 . The method of claim 2 , wherein the dimensions of the channel include a width of the channel.

4 . The method of claim 1 , wherein the microfluidic device further comprises a sequestration pen and the automatically-identified assay area AA x is further identified based, at least in part, on the dimensions of the sequestration pen.

5 . The method of claim 4 , wherein the dimensions of the sequestration pen include a width of the sequestration pen and a length of the sequestration pen.

6 . The method of claim 4 , wherein the automatically-identified assay area AA x is identified based, at least in part, on a position of a cell within the sequestration pen.

7 . The method of claim 6 , further comprising detecting the position of the cell within the sequestration pen.

8 . The method of claim 1 , wherein the automatically-identified assay area AA x is identified based, at least in part, on whether the assay is a secretion assay.

9 . The method of claim 1 , wherein the automatically-identified assay area AA x is identified based, at least in part, on the properties of a reagent or analyte used in the assay.

10 . The method of claim 4 , wherein the automatically-identified assay area AA x is identified based, at least in part, on the location of a reagent or analyte that has been affixed to a portion of the sequestration pen or a portion of the channel.

11 . The method of claim 10 , further comprising affixing the reagent or analyte to the portion of the sequestration pen by using structured light to actuate solidification of a light-actuated polymer network.

12 . The method of claim 1 , wherein digital images of said set of digital images I i (i=1 to n) are collected periodically.

13 . The method of claim 12 , wherein the period for collecting digital images of said set of digital images I i (i=1 to n) is once every 3 to 5 minutes.

14 . The method of claim 1 , wherein the set of digital images I i (i=1 to n) comprises a set of pixels P i,j and wherein calculating a rate of change Δ x further comprises determining a light intensity value L i,j of the set of pixels P i,j or a subset of the set of pixels P i,j .

15 . The method of claim 14 , wherein determining a light intensity value L i,j for a pixel P i,j comprises subtracting a background level of light intensity from the observed level of light intensity (L i,j (observed)−L i,j (background)).

16 . The method of claim 15 , wherein L i,j (background) is the light intensity value measured for the same pixel P j at the beginning of the assay (t=0) or just prior to the start of the assay.

17 . The method of claim 15 , wherein L i,j (background) is a light intensity value L ctrl observed for a control area.

18 . The method of claim 1 , wherein rate of change Δ x is a vector or other mathematical expression that represents the rate of change of two or more of the parameters selected from the group consisting of L i,avg , σ i , L i,min , and L i,max .

19 . The method of claim 1 , wherein the threshold 4° is based on the Δ avg and the standard deviation σ° for the rates of change Δ x corresponding to k different assay areas AA x (x=1 to k).

20 . The method of claim 19 , wherein the threshold Δ° is equal to Δ avg +1.6σ°.

21 . A machine readable storage device for storing non-transitory machine readable instructions for carrying out the methods of any one of claims 1 to 20 .

22 . An automated method of detecting a quantity of an analyte within a microfluidic device comprising one or more circuit elements, the method comprising:
collecting a set of digital images I i (i=1 to n) of an automatically-identified assay area AA x , wherein the automatically-identified assay area AA x , is identified based, at least in part, on the dimensions of the one or more circuit elements; calculating a rate of change Δ x over the course of all or part of the assay based on the set of digital images I i of the automatically-identified assay area AA x ; and determining a quantity of an analyte associated with the rate of change Δ x based on a calibration curve, wherein the calibration curve comprises one or more rates of change Δ x corresponding to known concentrations of the analyte.

23 . The method of claim 22 , further comprising determining the one or more rates of change Δ x corresponding to known concentrations of the analyte.

24 . The method of claim 23 , further comprising:
collecting one or more sets of digital images I i (i=1 to n) of one or more automatically-identified assay areas AA x associated with known concentrations of the analyte; and calculating the one or more rate of change Δ x over the course of all or part of the assay based on the one or more set of digital images I i of the automatically-identified assay area AA y .

25 . A machine readable storage device for storing non-transitory machine readable instructions for carrying out the methods of any one of claims 22 to 24 .

Assignees

• Berkeley Lights Inc

Inventors

• Du Fenglei

Classifications

• G01N21/8483

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