Analysis systems with microfluidic devices, microfluidic devices and related methods

Patent metadata

Field	Value
Publication number	US-12103003-B2
Application number	US-201917048153-A
Country	US
Kind code	B2
Filing date	Nov 5, 2019
Priority date	Nov 13, 2018
Publication date	Oct 1, 2024
Grant date	Oct 1, 2024

Abstract

Analysis systems with a housing having a chamber sized and configured to receive at least one microfluidic device. The systems also include an optic system coupled to the housing in optical communication with the at least one microfluidic device, a controller coupled to the optic system, a heat source coupled to the optic system and thermally coupled to the at least one microfluidic device held in the housing, and a sub-array selection module in communication with the controller. The sub-array selection module is configured to select a sub-set of sets of microwells of at least one fluid channel of the microfluidic device for imaging by the optic system after a reaction step (e.g., one thermocycle) during an assay.

First claim

That which is claimed is:

1. A method of identifying a target species and/or a target molecule in a microfluidic device configured to conduct PCR reactions in a plurality of sets of microwells, comprising:
exciting a defined sub-array of the plurality of sets of microwells in the microfluidic device, wherein the microfluidic device comprises at least a first fluid channel with some of the plurality of sets of microwells positioned along the first fluid channel;
obtaining signal intensity data from only the defined sub-array of the plurality of sets of microwells, wherein the obtaining signal intensity data comprises obtaining an analog signal that can provide real-time PCR data as amplitude changes versus a PCR cycle number for an input material in the plurality of sets of microwells and concentration of molecules of a defined type is about, equal to, or greater than one molecule per microwell, and wherein the analog signal defines a threshold cycle or cycle threshold, Ct, that identifies a microwell reaction as positive and a number of target molecules/concentration of the target molecule for a target species and/or type of molecule when fluorescence signal intensity (Si) is greater than a threshold value or negative if the fluorescence signal intensity (Si) is always less than the threshold value for a given number of PCR cycles;
scanning part or all of the plurality of sets of microwells after, before, or before and after the PCR reactions to obtain images of the plurality of sets of microwells for identifying positive and negative PCR reactions associated with digital PCR; and
electronically identifying microwells in the plurality of sets of microwells that are positive for one or more target analyte molecules.

2. The method of claim 1 , further comprising, fluidly loading a bead slurry pre-exposed to a respective sample for analysis, then sealing the plurality of sets of microwells along the first fluid channel before the obtaining step so that after sealing, each microwell of the plurality of sets of microwells is in fluid isolation from the others, wherein the plurality of sets of microwells along the first fluid channel are in fluid communication only during the loading, prior to the sealing step.

3. The method of claim 1 , further comprising changing the defined sub-array of the plurality of sets of microwells to a different defined sub-array after each of a plurality of successive reaction steps of an assay whereby some microwells of the plurality of sets of microwells are not imaged after each reaction step.

4. The method of claim 1 , wherein the microfluidic device further comprises:
a second fluid channel with a plurality of sets of microwells positioned along the second fluid channel;
a third fluid channel with a plurality of sets of microwells positioned along the third fluid channel; and
a fourth second fluid channel with a plurality of sets of microwells positioned along the fourth fluid channel,
wherein a first set of microwells of the plurality of sets of microwells of each of the first, second, third and fourth fluid channels are aligned in a first row, wherein a second set of microwells of the plurality of sets of microwells of each of the first, second, third and fourth fluid channels are aligned in a second row, wherein a third set of microwells of the plurality of sets of microwells of each of the first, second, third and fourth fluid channels are aligned in a third row, and wherein a fourth set of microwells of the plurality of sets of microwells of each of the first, second, third and fourth fluid channels are aligned in a fourth row.

5. The method of claim 4 , wherein the first, second, third and fourth fluid channels comprise straight or arcuate segments that are substantially parallel with each other.

6. The method of claim 4 , wherein the defined sub-array of the plurality of sets of microwells is a single one of the first, second, third and fourth rows.

7. The method of claim 1 , wherein the microfluidic device further comprises a second fluid channel adjacent to the first fluid channel.

8. The method of claim 1 , wherein the microfluidic device is configured with a plurality of spaced apart fluid channels, with the first fluid channel being one of the plurality of spaced apart fluid channels, wherein each of the plurality of spaced apart fluid channels comprises a plurality of sets of microwells, wherein the fluid channels are in fluid isolation.

9. The method of claim 8 , wherein the defined sub-array of the plurality of sets of microwells reside in at least two adjacent fluid channels of the plurality of spaced apart fluid channels.

10. The method of claim 1 , further comprising electronically identifying a location of one or more sets of microwells of the plurality of sets of microwells residing in one or more positions in the microfluidic device before the exciting and obtaining steps and defining positions of others of the sets of microwells based at least in part on the identified location.

11. The method of claim 1 , further comprising before the obtaining step, and before the exciting, selecting a filter to provide an encoding wavelength for the exciting step.

12. The method of claim 1 , wherein the Ct is a cycle number where Si>>Bs, where >>is at least a 5-10% greater than Bs and/or 5-10 times a standard deviation of Bs, and where Bs is background fluorescence signal.

13. The method of claim 12 , wherein >>is a factor of two greater than Bs.

14. The method of claim 12 , where Bs is background fluorescence signal as measured at a PCR negative reaction.

15. The method of claim 1 , wherein the microfluidic device comprises a plurality of fluid channels with the first fluid channel being one of the plurality of fluid channels, wherein the plurality of fluid channels are arranged with respective microwells thereof aligned in different rows, wherein the analog signal is obtained for each defined sub-array of the plurality of sets of microwells in one row that changes after every other or each of a plurality of different reaction steps of an assay.

16. The method of claim 15 , wherein each defined sub-array of the plurality of sets of microwells comprises two sets of microwells.

17. The method of claim 1 , wherein the analog signal comprises an average, median, mode, or weighted value of Si.

18. The method of claim 1 , wherein the obtained signal intensity data is carried out by using a camera with a field of view (FOV) that covers only a defined sub-array of the plurality of sets of microwells of the microfluidic device at any one time.

19. The method of claim 1 , wherein the microfluidic device with the first fluid channel with the plurality of sets of microwells spaced apart along the first fluid channel comprises a plurality of additional fluid channels, each comprising a respective plurality of sets of microwells spaced apart along its respective length, and wherein the microfluidic device further comprises a separate material input port for each of the first and the plurality of additional fluid channels and at least some of the fluid channels share a common opposing port, the method further comprising before the obtaining step:
fluidly loading a bead slurry pre-exposed to a respective sample for analysis into the respective input ports;
magnetically directing the bead slurries to enter into different sets of the plurality of sets of microwells along the fluid channels;
flowing a fluid master mix comprising a dye from the opposing port into the fluid channels toward the material input ports; and then

Assignees

• Univ North Carolina Chapel Hill

Inventors

• Henley William Hampton
• Pfefferle Adam D
• Ramsey John Michael
• Perkowski Ellen Foot

Classifications

• C12M1/00

  Apparatus for enzymology or microbiology · CPC title

• C12M1/02

  with agitation means; with heat exchange means · CPC title

• C12M1/36

  including condition or time responsive control, e.g. automatically controlled fermentors (controlling or regulating in general G05) · CPC title

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• C12M1/34 Primary

  Measuring or testing with condition measuring or sensing means, e.g. colony counters · CPC title