Single cell microfluidic device

Patent metadata

Field	Value
Publication number	US-10526595-B2
Application number	US-201815950982-A
Country	US
Kind code	B2
Filing date	Apr 11, 2018
Priority date	Oct 14, 2015
Publication date	Jan 7, 2020
Grant date	Jan 7, 2020

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 micro fluidic device configured to non-destructively obtaining a genotype
of a plant cell, the device comprising:
an inlet configured to receive a plant cell sample comprising a plurality of plant cells;
a micro fluidic channel in fluid communication with the inlet;
an obstacle array configured to focus the plant cell sample to a sidewall of the microfluidic channel;
an electrode array configured to separate a first constituent from the focused plant cell sample by deflecting the first constituent of the focused plant cell sample towards a central portion of the micro fluidic channel;
a single-cell trapping array located downstream from the electrode array, the single-cell trapping array comprising a microwell array configured to trap a single plant cell from the first constituent of the plant cell sample in a microwell of the microwell array;
a polymer membrane encapsulating the single-cell trapping array; and
an external micro-manipulating instrument configured to pierce the polymer membrane and penetrate a respective cell wall of the trapped single plant cell to extract mRNA from the trapped single plant cell using a dielectrophoresis (DEP) force under an alternating current (AC) field applied to the external micro-manipulating instrument.

2. The device of claim 1 , wherein the external micro-manipulating instrument comprises a probe tip of an Atomic Force Microscope (AFM).

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

4. The device of claim 1 , wherein the polymer membrane has a thickness less than 5 micron.

5. The device of claim 1 , wherein the polymer membrane is configured to be resealable after removal of the external micro-manipulating instrument.

6. The device of claim 1 , wherein the plant cell sample is a plant selected from maize or canola.

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

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

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

10. The device of claim 9 , wherein the plurality of plant cells are tetrad mlcrospores.

11. The device of claim 1 , further comprising an analysis chamber configured to analyze mRNA extracted from the trapped single plant cell to determine gene expression patterns of the plant cell.

12. The device of claim 1 , further comprising an analysis chamber configured to determine the genotype of the trapped single plant cell from cDNA obtained from mRNA extracted from the trapped single plant cell.

Assignees

• Univ California
• Pioneer Hi Bred Int

Inventors

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

Classifications

• C12Q2565/107

  Alteration in the property of hybridised versus free label oligonucleotides · CPC title

• C12N15/101

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

• B01L3/502753

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

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title