Automated identification of assay areas in a microfluidic device and detection of assay positive areas based on rate of change of image light intensity

Patent metadata

Field	Value
Publication number	US-10578630-B2
Application number	US-201514964025-A
Country	US
Kind code	B2
Filing date	Dec 9, 2015
Priority date	Dec 9, 2014
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

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

What is claimed:

1. A method of detecting an assay-positive assay area within a microfluidic device comprising a microfluidic circuit having a sequestration pen and a flow channel configured to contain a fluidic medium, the method comprising automatically:
flowing a fluidic medium comprising a micro-object into the flow channel of the microfluidic device;
identifying an assay area based at least in part on a structural feature of the microfluidic circuit of the microfluidic device, wherein the structural feature is an opening from the sequestration pen to the flow channel;
collecting a set of digital images of the assay area;
calculating a rate of change of at least one parameter that characterizes a distribution of a set of light intensity values over the course of all or part of the assay based on the set of digital images of the identified assay area;
comparing the rate of change to a threshold value related to the at least one parameter; and
determining that the identified assay area is assay-positive if the rate of change is greater than the threshold value.

2. The method of claim 1 , wherein the assay area is identified based at least in part on a dimension of the channel.

3. The method of claim 2 , wherein the dimension of the channel includes a width or length of the channel.

4. The method of claim 1 , wherein the assay area is identified based at least in part on a dimension of the sequestration pen.

5. The method of claim 4 , wherein the dimension of the sequestration pen includes a width or a length of the sequestration pen.

6. The method of claim 1 , wherein identifying the assay area further comprises determining a position of a biological micro-object within the sequestration pen.

7. The method of claim 1 , wherein the assay area is identified based at least in part on whether the assay is a secretion assay or an antigen detection assay.

8. The method of claim 7 , wherein the assay is a secretion assay and comprises a plurality of micro-objects disposed in the assay region area.

9. The method of claim 8 , wherein the assay area is further configured to exclude regions of the assay area that represent abberant or outlier data.

10. The method of claim 8 , wherein the assay augments or extends the automatically-identified assay areas to include additional pixels, dependent on the at least one parameter.

11. The method of claim 8 , wherein the micro-objects comprise beads and an antigen-specific binding agent on the beads, and wherein the micro-objects are disposed in the flow channel and/or in a portion of the sequestration pen proximal to the flow channel.

12. The method of claim 11 , further comprising flowing in a labeled antigen-binding agent with the micro-objects, wherein the labeled antigen-binding agent is configured to bind to a different portion of the antigen of interest than the antigen-specific binding agent on the beads.

13. The method of claim 1 , wherein the assay area is identified based at least in part on a diffusion correlated property of a reagent or analyte used in the assay.

14. The method of claim 1 , wherein the assay area 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.

15. The method of claim 14 , further comprising affixing the reagent or analyte to the portion of the sequestration pen or the portion of the channel by using structured light to solidify a polymer network.

16. The method of claim 1 , wherein the digital images are collected over time comprising a first time point and a second time point and with periodicity.

17. The method of claim 16 , wherein a digital image of the set is collected every 3 to 5 minutes.

18. The method of claim 1 , wherein each digital image of the set of digital images comprises a set of pixels, and wherein calculating the rate of change comprises determining a light intensity value or a set of light intensity values for the set of pixels or a subset of the set of pixels determined based on the identified assay area.

19. The method of claim 18 , wherein determining a light intensity value for a pixel comprises subtracting a background level of light intensity from an observed level of light intensity.

20. The method of claim 19 , wherein the background level of light intensity is measured for a given pixel prior to the start of the assay or at the beginning of the assay.

21. The method of claim 20 , wherein the background level of light intensity is a light intensity value observed for a control area of the microfluidic device.

22. The method of claim 1 , wherein the at least one parameter that characterizes the distribution of light intensity values comprises one or more of: the average light intensity value, the standard deviation of the light intensity value, a minimum light intensity value, and a maximum light intensity value determined for a set of pixels collected in each of the images in the set of images.

23. The method of claim 1 , further comprising identifying a plurality of assay areas and wherein each image of the set of digital images shows a plurality of assay areas, and the threshold value for the at least one parameter comprises a sum of (i) an average rate of change for the distribution of values for the at least one parameter, wherein the distribution is obtained from the plurality of assay areas, and (ii) a standard deviation for the distribution of values obtained for each of the plurality of assay areas.

24. The method of claim 23 , wherein the threshold is equal to the average rate of change plus 1.6 times the standard deviation for the distribution of the rates of change.

25. A non-transitory machine readable storage device for storing non-transitory machine readable instructions for carrying out the method of claim 1 .

26. The method of claim 1 , wherein the sequestration pen comprises an isolation region and a connection region configured to fluidically connect the isolation region to the flow channel.

27. The method of claim 1 , wherein the microfluidic device comprises a plurality of sequestration pens and a plurality of corresponding assay areas.

28. The method of claim 1 , wherein the sequestration pen opens laterally to the flow channel and the lateral orientation is configured to modulate exchange between fluid in the flow channel and the sequestration pen.

29. The method of claim 1 , wherein the assay area is identified based on one or more of: the velocity of the fluidic medium, the viscosity of the fluidic medium, the presence of a polymerization agent within the fluidic medium, physical and chemical properties of analytes measured in the assay, the physical location of reagents or analytes used in the assay, the number of cells being assayed, or the amount of noise and background produced by analytes and/or reagents used in the assay.

Assignees

• Berkeley Lights Inc

Inventors

• Du Fenglei

Classifications

• G01N2021/755

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• B01L2200/148

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