MICROFLUIDIC DEVICE POSSESSING STRUCTURES ENABLING DIFFERENTIAL ANALYSIS OF A SINGLE CELL'S CONSTITUENTS

Abstract

A method and a micro fluidic device comprising at least one micro fluidic structure for differential extraction of nuclear and extra-nuclear constituents of a single cell, said micro fluidic structure comprising a feeding channel for receiving a volume of a sample containing at least one cell, at least one trapping structure for capturing a single cell, and at least one output channel in fluid connection with the at least one trapping structure, wherein the at least one trapping structure extends from one side of the feeding channel substantially perpendicular to longitudinal axis of the feeding channel, the at least one trapping structure possessing an aperture at its end opposite to the fluid channel and in fluid communication with an output channel, said aperture being configured to provide a narrow section such that the nucleus of a cell captured in the trapping structure cannot pass through said narrow section into the output channel.

Claims

1. A microfluidic device comprising at least one microfluidic structure for differential extraction of nuclear and extra-nuclear constituents of a single cell, said microfluidic structure comprising: a feeding channel for receiving a volume of a sample containing at least one cell, at least one trapping structure for capturing a single cell, and at least one outlet channel in fluid connection with the at least one trapping structure, wherein the at least one trapping structure extends from one side of the feeding channel substantially perpendicular to longitudinal axis of the feeding channel, the at least one trapping structure possessing an aperture at its end opposite to the fluid channel and in fluid communication with an outlet channel, said aperture being configured to provide a narrow section such that the nucleus of a cell captured in the trapping structure cannot pass through said narrow section into the outlet channel.

2. The microfluidic device according to claim 1, further comprising at least one buffer channel in fluid connection with the feeding channel, wherein the at least one buffer channel converges with the feeding channel at the side of the feeding channel opposite to the at least one trapping structure, andwith respect to the direction of flow within the feeding channelat a position along the feeding channel preceding the position of the at least one trapping structure.

3. The microfluidic device according to claim 1, comprising two or more buffer channels.

4. The microfluidic device according to claim 1, wherein the at least one buffer channel or the two or more buffer channel converge(s) with the feeding channel in an angle of less than 90, preferably in an angle in the range of about 30 to about 70, more preferably in an angle in the range of about 40 to about 60, and most preferably in an angle in the range of about 45 to about 55.

5. The microfluidic device according to claim 1, wherein the narrow section has in inner diameter in the range of about 1 m to about 4 m.

6. The microfluidic device according to claim 1, wherein the outlet channel comprises two or more legs.

7. The microfluidic device according to claim 1, wherein the outlet channel or the legs of the outlet channel is/are is in fluid connection with at least one auxiliary chamber for detecting and/or analyzing at least one constituent of the cell.

8. The microfluidic device according to claim 1, wherein the microfluidic structure comprises at least one valve for directing the flow of fluid within the microfluidic structure.

9. The microfluidic device according to claim 8, wherein the inlet and/or the outlet of the feeding channel, the inlet and/or outlet of the at least one buffer channel, the inlet and/or outlet(s) of the outlet channel and/or the diversion within the outlet channel to the legs comprise the valve.

10. A method of manufacturing a microfluidic device as defined in claim 9, wherein the microfluidic structure is produced by injection molding a polymer, and subsequently sealing the channels by bonding a polymer film to the molded structure.

11. Use of a microfluidic device according to claim 9 for differentially extracting nuclear and extra-nuclear constituents of a cell.

12. The use according to claim 11, wherein the nuclear and/or extra-nuclear constituents are nucleic acid molecules.

13. A method for differentially extracting nuclear and extra-nuclear constituents of a single cell, the method comprising the steps of: providing at least one cell to the feeding channel of a microfluidic device according to claim 9; capturing the at least one cell in the at least one trapping structure; lysing the cell captured in the at least one trapping structure without affecting integrity of the cell's nucleus by supplying a first lysis buffer to the cell; releasing the extra-nuclear constituents of the cell into the outlet channel; transferring the extra-nuclear constituents of the cell from the outlet channel into an auxiliary chamber for further processing; lysing the cell's nucleus by supplying a second lysis buffer to the nucleus; releasing the constituents of the cell's nucleus into the outlet channel; and transferring the constituents of the cell's nucleus from the outlet channel to an auxiliary chamber for further processing.

14. The method according to claim 13, further comprising: amplification of at least one nucleic acid sequence of the cell's nuclear constituents; and amplification of at least one nucleic acid sequence of the cell's extra-nuclear constituents.

15. The method according to claim 14, further comprises analyzing the nucleotide sequence of the amplification product of the at least one nucleic acid sequence of the cell's nuclear constituents.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. Such embodiment does not necessarily represent the full scope of the invention, however, and reference is made therefore to the claims and herein for interpreting the scope of the invention.

[0097] In the drawings:

[0098] FIG. 1 shows a schematic illustration of an embodiment of a micro fluidic structure in accordance with the invention.

[0099] FIG. 2 shows a schematic illustration of another embodiment of a microfluidic structure in accordance with the invention.

[0100] FIG. 3A and FIG. 3B display graphs illustrating the amplification of nucleic acid sequenced of a single cell isolated by a method according to the invention.

[0101] FIG. 4 displays a cross sectional view of a channel in a preferred embodiment of the microfluidic device.

DETAILED DESCRIPTION OF EMBODIMENTS

[0102] Referring to FIG. 1, a schematic illustration of an embodiment of a microfluidic structure in accordance with the invention is shown. The microfluidic structure 1 comprises a feeding channel 2 possessing an inlet (cell inlet) 21 and a waste outlet (22). The microfluidic structure 1 comprises a trapping structure 3 in fluid communication with and orthogonally extending from the flow path of the feeding channel 2. The trapping structure 3 comprises an outlet 31 in fluid connection with an output channel 4. The fluid connection 34 between the trapping structure 3 and the output channel 4 provides a narrow section configured to prevent a cell 8 being captured in the trapping structure 3 from accessing the output channel 4. The output channel 4 possesses an outlet 42 which is or may get fluid connection with an auxiliary chamber which is configured for detecting and/or analyzing one or more cell constituents. The microfluidic structure 1 further comprises two buffer channels, a first buffer channel 5 and a second buffer channel 6. The first buffer channel 5 being in fluid communication with a first buffer reservoir 51, and the second buffer channel 6 in fluid communication with a second buffer reservoir 61. Optionally one of the first buffer reservoir 51 and the second buffer reservoir 61 contains a fluid maintaining integrity and viability of cells, whereas the other buffer reservoir contains a lysis buffer for lysing a cell captured in the trapping structure 3.

[0103] During operation, a flow of buffer or medium is provided via at least one of the buffer channels 5, 6 as indicated by the solid arrows. A cell migrating along the feeding channel 2 is forced within the feeding channel 2 towards the side opposite of the outlet 62 of the buffer channel 5 and/or 6 to be captured by the trapping structure 3 also located at the side of the feeding channel opposite to the outlets 52, 62 of the buffer channels 5, 6.

[0104] Referring to FIG. 2, another embodiment of the microfluidic structure according to the invention is schematically shown. The trapping device 33 has a wedge-shaped form provided that the trapping structure has a rectangular or square cross section. The microfluidic structure 10 comprises an output channel having a first leg 43 and a second leg 44, wherein the first leg 43 possesses an outlet 431 which is or may become in fluid communication with a first auxiliary chamber, and wherein the second leg 44 possesses an outlet 441 which is or may become in fluid communication with a second auxiliary chamber.

[0105] The output channel of the embodiment shown in FIG. 2 further comprises an actuatable two-way valve 7 for directing the flow coming from the trapping structure to one of the two legs 43, 44 of the output channel.

[0106] Referring to FIG. 3A a graph is shows visualizing the results of amplifications of a fragment of -actin mRNA of a single cell captured by using a microfluidic device according to the invention. The mRNA of the cell was obtained by the method according to the present invention. The fragment was amplified in a real-time PCR using specific primers after reverse transcription of the cell's mRNA using an oligo-dT-Primer. Increase of fluorescence upon the amplification cycles were monitored for the cell's mRNA (dashed line) and from an amount of mRNA equivalent to 3.5 cells (solid line) as positive control. A negative control without mRNA did not lead to any detectable fluorescence.

[0107] Referring to FIG. 3B a graph is shown which visualizes the results of amplifications of RNAse P DNA. The genomic DNA was obtained from the same single cell as the mRNA used in the amplification reaction shown in FIG. 3A. The genomic DNA was first subjected to whole genome amplification (WGA). The product of the WGA was diluted to enable real-time PCR amplification of a fragment of the RNase P gene. Increase of fluorescence upon the amplification cycles were monitored for the genomic DNA of the single cell (dashed line) and for 2 ng genomic DNA as positive control (solid line). A negative control without DNA did not lead to any detectable fluorescence.

[0108] FIG. 4 illustrates a preferred configuration of the channels within an embodiment of the microfluidic device. A cross sectional view of a region of a microfluidic structure 70 comprising a channel 73 is schematically presented. The microfluidic structure 70 comprises a base 71, i.e. a polymeric one-layer device comprising the channel 73, and a lid 72 for sealing the channel 73. The lid 72 may be a polymer film. The channel 73 may be any channel of the micro fluidic structure such as the feeding channel, the buffer channel(s) and/or the output channel (including any legs thereof). The channel 73 is delimitated by its bottom 74, its ceiling 75 and its side walls 76, 77. The angle denotes the angle by which the slope of the side wall deviates from the perpendicular plane with respect to the plane of the bottom of the channel. The angle between wall 76 (representing the hypotenuse) of channel 73 and a perpendicular dropped from the outer edge of the channel's bottom plane (the adjacent cathetus) may in the range of between about 3 to about 10.

TABLE-US-00001 REFERENCE SYMBOL LIST 1 microfluidic structure 2 feeding channel 3 trapping structure 4 output channel 5 first buffer channel 6 second buffer channel 7 valve 8 cell 10 microfluidic structure 21 cell inlet 22 waste outlet 31 outlet 33 trapping structure 34 narrow section 42 outlet 43 leg 44 leg 51 buffer reservoir 52 outlet 61 buffer reservoir 62 outlet 70 microfluidic structure 71 base 72 lid 73 channel 74 bottom 75 ceiling 76 side wall 77 side wall 431 outlet 441 outlet