Single cell microfluidic device

Patent metadata

Field	Value
Publication number	US-9862941-B2
Application number	US-201615288969-A
Country	US
Kind code	B2
Filing date	Oct 7, 2016
Priority date	Oct 14, 2015
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

Abstract

Methods of non-destructively obtaining the genotype of a plant cell from a plant sample are disclosed. The plant cell is isolated from a plant cell sample using an integrated microfluidic device. The integrated microfluidic device includes an individual cell trap located downstream from a microfluidic channel and, the microfluidic device is configured to trap the plant cell in the individual cell trap. mRNA is extracted from the plant cell by contacting the plant cell with an atomic force microscope (AFM) probe and by attracting mRNA from loci of interest to the probe end using a dielectrophoresis DEP force under the alternating current (AC) field applied to the probe. The genotype of the plant cell is determined from cDNA obtained from the extracted mRNA. Alternatively, the mRNA is analyzed to determine gene expression patterns of the plant cell.

First claim

What is claimed is:

1. A method of non-destructively obtaining a genotype of a plant cell, the method comprising:
(a) providing a plant cell sample comprising a plurality of plant cells;
(b) isolating a single plant cell using an integrated microfluidic device, wherein the integrated microfluidic device comprises a microfluidic channel having an inlet through which the plurality of plant cells are introduced into the device, wherein the integrated microfluidic device further comprises an obstacle array, an electrode array configured to transport the cells to a trapping region located downstream from the electrode array, wherein the trapping region further comprises a microwell array configured as a single-cell trapping array to capture the single plant cell in the microwell of the microwell array,
(c) extracting mRNA from the plant cell by contacting the plant cell with an external micro-manipulating instrument and by attracting mRNA from loci of interest to the micro-manipulating instrument using a dielectrophoresis (DEP) force under the alternating current (AC) field applied to the micro-manipulating instrument; and
(d) determining the genotype of the plant cell from cDNA obtained from the extracted mRNA.

2. The method of claim 1 , wherein dielectrophoresis is used to separate living plant cells from dead plant cells either prior to or during step (b).

3. The method of claim 1 , further comprising selecting the plant cell from step (d) and retrieving the selected plant cell.

4. The method of claim 1 , wherein the plurality of plant cells are selected from the group consisting of microspores and protoplasts.

5. The method of claim 1 , further comprising focusing the introduced plant cell sample towards a sidewall of the microfluidic channel.

6. The method of claim 5 , further comprising separating a first constituent from the focused plant cell sample.

7. The method of claim 6 , wherein the first constituent comprises live plant cells.

8. The method of claim 7 , wherein separating the first constituent from the focused plant cell sample comprises deflecting the first constituent of the focused plant cell sample towards a central portion of the microfluidic channel.

9. The method of claim 6 , further comprising fluidically transporting the first constituent through the microfluidic channel to the trapping region.

10. The method of claim 1 , wherein each plant cell is probed and said probing comprises:
disposing an Atomic Force Microscope (AFM) probe over the plant cell trapped in the single-cell trapping array; and
activating the probe to pierce a polymer membrane encapsulating the single-cell trapping array and penetrate a respective cell wall of the trapped plant cell.

11. The method of claim 10 , wherein the external micro-manipulating instrument comprises a probe tip of an AFM.

12. The method of claim 10 , wherein the polymer membrane has a thickness less than 5 micron.

13. The method of claim 12 , wherein the polymer membrane is configured to be resealable after removal of the external micro-manipulating instrument.

14. The method of claim 1 , wherein the external micro-manipulating instrument comprises a dielectrophoretic nano tweezer (DENT).

15. The method of claim 1 , wherein the plant cell is from a plant selected from maize or canola.

16. The method of claim 1 , wherein the plant cell is a microspore having a preselected genotype wherein the preselection is based upon one allele or multiple alleles.

17. A method of non-destructively obtaining gene expression patterns of a plant cell, the method comprising:
(a) providing a plant cell sample comprising a plurality of plant cells;
(b) isolating a single plant cell using an integrated microfluidic device, wherein the integrated microfluidic device comprises a microfluidic channel having an inlet through which the plurality of plant cells are introduced into the device, wherein the integrated microfluidic device further comprises an obstacle array, an electrode array configured to transport the live cells to a trapping region located downstream from the electrode array, wherein the trapping region further comprises a microwell array configured as a single-cell trapping array to capture the live single plant cell in the microwell of the microwell array,
(c) extracting mRNA from the plant cell by contacting the plant cell with an external micro-manipulating instrument and by attracting mRNA from loci of interest to the micro-manipulating instrument using a dielectrophoresis (DEP) force under the alternating current (AC) field applied to the micro-manipulating instrument; and
(d) analyzing the mRNA to determine gene expression patterns of the plant cell.

18. The method of claim 17 , wherein mRNA is analyzed using qRT-PCR.

19. The method of claim 17 , wherein dielectrophoresis is used to separate living plant cells from dead plant cells either prior to or during step (b).

20. The method of claim 17 , further comprising selecting the plant cell with desired gene expression patterns of step (d) and retrieving the selected plant cell.

21. The method of claim 17 , wherein the plurality of plant cells is are selected from the group consisting of microspores and protoplasts.

22. The method of claim 17 , wherein the plurality of plant cells are tetrad microspores.

23. The method of claim 17 , further comprising focusing the introduced plant cell sample towards a sidewall of the microfluidic channel.

24. The method of claim 23 , further comprising separating a first constituent from the focused plant cell sample.

25. The method of claim 24 , wherein the first constituent comprises live plant cells.

26. The method of claim 24 , wherein separating the first constituent from the focused plant cell sample comprises deflecting the first constituent of the focused plant cell sample towards a central portion of the microfluidic channel.

27. The method of claim 24 , further comprising fluidically transporting the first constituent through the microfluidic channel to the trapping region.

28. The method of claim 17 , wherein each plant cell is probed and said probing comprises:
disposing the external micro-manipulating instrument over the plant cell trapped in the single-cell trapping array; and
activating the external micro-manipulating instrument to pierce a polymer membrane encapsulating the single-cell trapping array and penetrate a respective cell wall of the trapped plant cell.

29. The method of claim 28 , wherein the external micro-manipulating instrument comprises a probe tip of an AFM.

30. The method of claim 28 , wherein the polymer membrane has a thickness less than 5 micron.

31. The method of claim 30 , wherein the polymer membrane is configured to be resealable after removal of the external micro-manipulating instrument.

32. The method of claim 17 , wherein the external micro-manipulating instrument comprises a DENT.

33. The method of claim 17 , wherein the plant cell is from a plant selected from maize or canola.

Assignees

• Univ California
• Pioneer Hi Bred Int

Inventors

• Lee Abraham P
• Lee Do-Hyun
• Wickramasinghe H Kumar
• Tao Yinglei
• Li Xuan
• Yun Yue

Classifications

• B01L3/502753

  characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation · CPC title

• C12N15/1017

  by filtration, e.g. using filters, frits, membranes · CPC title

• B01L2300/0681

  Filter · CPC title

• C12N15/101

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title