Microfluidic PCR device

• US 9,777,317 B2
• Filed: 08/01/2013
• Issued: 10/03/2017
• Est. Priority Date: 08/03/2012
• Status: Active Grant

• Current Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS), STMicroelectronics SRL (STMicroelectronics NV)
• Original Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS), STMicroelectronics SRL (STMicroelectronics NV)
• Inventors: Spoto, Giuseppe Emanuele, Occhipinti, Luigi Giuseppe, Dall'Oglio, Cristian, Renna, Crocifisso Marco Antonio, Drazek, Laurent
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US13/956,677
• Publication Number:
  US 20140038193A1
• Time in Patent Office: 1,524 Days
• Field of Search: None
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

A microfluidic device (1000-1005), comprising: a semiconductor body (2) having a first side (2a) and a second side (2b) opposite to one another, and housing, at the first side, a plurality of wells (4), having a first depth; an inlet region (30) forming an entrance point for a fluid to be supplied to the wells; a main channel (6a) fluidically connected to the inlet region, and having a second depth; and a plurality of secondary channels (6b) fluidically connecting the main channel to a respective well, and having a third depth. The first depth is higher than the second depth, which in turn is higher than the third depth. According to an aspect, the microfluidic device further comprises a cover layer (8), arranged above the first side of the semiconductor body, configured for sealing the wells and provided with at least a first valve hole (54) which extends through the cover layer and overlaps, at least partially, the secondary channels; and a flexible layer (14), arranged above the cover layer and provided with at least a protrusion (74) extending through the first valve hole towards the semiconductor body and overlapping, at least partially, the secondary channels, the flexible layer being configured such that, when a pressure is applied on it, the protrusion contacts the semiconductor body and enters the secondary channels thus fluidically isolating the wells from one another.

Claims

1. A microfluidic device, comprising:

a body having a first side and a second side opposite the first side, said first side having;

at least two amplification reaction chambers, the amplification reaction chambers configured to store reagents and samples;

an inlet region forming an entrance for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

at least two secondary channels, each one of the secondary channels fluidically connecting the main channel to a respective one of the amplification reaction chambers,

wherein the amplification reaction chambers extend within the body for a first depth, the main channel extends within the body for a second depth, the secondary channels extend within the body for a third depth, the first depth being greater than the second depth, and the second depth being greater than the third depth.

2. The microfluidic device according to claim 1, wherein the secondary channels are coupled to opposite sides of the main channel and are staggered with respect to one another.

3. The microfluidic device according to claim 1, wherein the main channel and the secondary channels are configured to transport the fluid within the main channel and the secondary channels by capillary action.

4. The microfluidic device according to claim 1, further comprising a flexible layer arranged above the first side of the body and provided with at least a protrusion extending towards the first side of the body and overlapping, at least partially, the secondary channels, the flexible layer being configured to selectively, fluidically isolate a first one of the amplification reaction chambers from a second one of the amplification reaction chambers, upon application of a pressure to the flexible layer.

5. The microfluidic device according to claim 4, further comprising a cover layer, arranged above the first side of the body and configured for sealing the amplification reaction chambers.

6. The microfluidic device according to claim 5, wherein the flexible layer is arranged above the cover layer, the cover layer being provided with at least a first valve hole which extends through the cover layer and overlaps, at least partially, the secondary channels, the protrusion extending through the first valve hole.

7. The microfluidic device according to claim 5, further comprising a plurality of air exhaust channels, each of the air exhaust channels being fluidically coupled to a respective one of the amplification reaction chambers, and configured to exhaust a gas from within the amplification reaction chambers to an environment external to the amplification reaction chambers.

8. The microfluidic device according to claim 7, wherein the air exhaust channels extend through the cover layer, the microfluidic device further comprising a membrane layer arranged above the cover layer in such a way to cover the air exhaust channels and configured to be impermeable to said fluid and permeable to said gas.

9. The microfluidic device according to claim 8, wherein the air exhaust channels further extend through the flexible layer, and wherein the membrane layer is arranged above the flexible layer.

10. The microfluidic device according to claim 1, wherein the inlet region is accessible through a pipette or micro pipette for supplying said fluid.

11. The microfluidic device according to claim 5, wherein the body houses one or more grooves extending at the first side, at least partially around one or more of the amplification reaction chambers, the cover layer being sealed to the body by glue within said grooves.

12. The microfluidic device according to claim 11, wherein the glue is a biocompatible, non-fluorescent, glue.

13. The microfluidic device according to claim 5, further comprising a first bi-adhesive layer arranged between the body and the cover layer, configured to couple the cover layer to the body.

14. The microfluidic device according to claim 13, wherein the first bi-adhesive layer is provided with at least two inspection holes, extending through the first bi-adhesive layer for its entire thickness, the at least two inspection holes being at least partially aligned with the at least two amplification reaction chambers, respectively.

15. The microfluidic device according to claim 1, further comprising an integrated heater formed at the second side of the body.

16. The microfluidic device according to claim 1, further comprising an integrated temperature sensor formed at the second side of the body.

17. The microfluidic device according to claim 4, forming a microreactor, wherein each one of the amplification reaction chambers accommodates an array of nucleic acid probes at respective locations configured to hybridize to respective target nucleic acids;

and wherein said fluid is a solution containing primers, capable of binding to target nucleic acids, nucleotides, nucleic acid extending enzymes and nucleic acids.

18. The microfluidic device according to claim 4, wherein the flexible layer is transparent to at least a first wavelength and a second wavelength, the first wavelength and the second wavelength different from one another.

19. The microfluidic device according to claim 5, wherein the cover layer is transparent to at least a first wavelength and a second wavelength, the first wavelength and the second wavelength different from one another.

20. The microfluidic device according to claim 1, further comprising an enclosing structure having lateral walls arranged on the first side of the body to completely encircle the amplification reaction chambers, said enclosing structure being further provided with a reservoir fluidically coupled to the inlet region and configured for containing an amount of said liquid greater than an amount of said liquid that can be contained by the inlet region.

30. A method of operating the microfluidic device of claim 4, comprising:

supplying a liquid solution at the inlet region; and

after the step of supplying, applying a pressure on the flexible layer such that the protrusion contacts the body and enters within at least one of the secondary channels, thus fluidically isolating a respective one of the at least two amplification reaction chambers from the main channel.

21. A microfluidic device, comprising:

a body having a first side and a second side opposite to one another, said first side having;

a plurality of amplification reaction chambers, the amplification reaction chambers configured to store reagents and samples;

an inlet region forming an entrance point for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

a plurality of secondary channels, each one of the secondary channels fluidically connecting the main channel to a respective one of the, the amplification reaction chambers;

a cover layer, arranged above the first side of the body, configured to seal the amplification reaction chambers and provide at least a first valve hole which extends through the cover layer and overlaps, at least partially, the secondary channels; and

a flexible layer, arranged above the cover layer and provided with at least a protrusion extending through the first valve hole towards the body and overlapping, at least partially, a respective one of the secondary channels, the flexible layer being configured such that, when a pressure is applied on the flexible layer, the protrusion contacts the body and enters within the respective one of the secondary channels, thus fluidically isolating a respective one of the plurality amplification reaction chambers from the main channel.

22. A cartridge comprising:

a microfluidic device including;

a body having a first side and a second side opposite the first side, said first side having;

at least two amplification reaction chambers, the amplification reaction chambers configured to store reagents and samples;

an inlet region forming an entrance for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

at least two secondary channels, each one of the secondary channels fluidically connecting the main channel to a respective one of the amplification reaction chambers,

wherein the amplification reaction chambers extend within the body for a first depth, the main channel extends within the body for a second depth, the secondary channels extend within the body for a third depth, the first depth being greater than the second depth, and the second depth being greater than the third depth; and

a device holder configured to house said microfluidic device, including a base portion and a cover portion, the cover portion having a protrusion, the base portion and the cover portion configured to be coupled to one another and to exert pressure on the microfluidic device along a direction parallel to a direction of extension of the protrusion, so that the protrusion contacts the body and enters within at least one of the secondary channels, thus fluidically isolating a respective one of the at least two amplification reaction chambers from the main channel.

35. The microfluidic device according to claim 22, wherein the main channel and the secondary channels are configured to transport the fluid within the main channel and the secondary channels by capillary action.

36. The microfluidic device according to claim 22, further comprising a flexible layer arranged above the first side of the body and provided with at least a protrusion extending towards the first side of the body and overlapping, at least partially, at least one of the at least two secondary channels, the flexible layer being configured to selectively, fluidically isolate a respective one of the at least two amplification reaction chambers from the main channel, upon application of a pressure to the flexible layer.

37. The microfluidic device according to claim 36, further comprising a cover layer, arranged above the first side of the body and configured for sealing the amplification reaction chambers.

38. The microfluidic device according to claim 37, wherein the flexible layer is arranged above the cover layer, the cover layer being provided with at least a first valve hole which extends through the cover layer and overlaps, at least partially, at least one of the at least two secondary channels, the protrusion extending through the first valve hole.

23. A nucleic acid amplification apparatus, comprising:

a microfluidic device that includes;

a body having a first side and a second side opposite the first side, said first side having;

at least two amplification reaction chambers, the amplification reaction chambers configured to store reagents and samples;

an inlet region forming an entrance for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

at least two secondary channels, each one of the secondary channels fluidically connecting the main channel to a respective one of the amplification reaction chambers,

wherein the amplification reaction chambers extend within the body for a first depth, the main channel extends within the body for a second depth, the secondary channels extend within the body for a third depth, the first depth being greater than the second depth, and the second depth being greater than the third depth.

24. The apparatus according to claim 23, comprising a temperature control module for cyclically controlling an operative temperature of the solution in accordance with a temperature profile.

25. The apparatus according to claim 23, further comprising a reader device and wherein the microfluidic device is mounted on a board to form a cartridge loadable into the reader device.

26. The apparatus according to claim 25, wherein the reader device is an optical reader and includes:

a light source for illuminating the microfluidic device with light at a first wavelength; and

an image detector, configured to receive light radiation at a second wavelength emitted by the microfluidic device, in response to the light at the first wavelength.

27. A method of manufacturing a microfluidic device, comprising:

forming at least two amplification reaction chambers for storing reagents and samples in a first side of a body that also includes a second side opposite to the first side, the at least two amplification reaction chambers having a first depth;

forming an inlet region in the first side, the inlet region forming an entrance for a fluid to be supplied to the amplification reaction chambers;

forming a main channel in the first side, the main channel being fluidically connected to the inlet region, and having a second depth; and

forming at least two secondary channels in the first side, each one of the at least two secondary channels fluidically connecting the main channel to a respective one of the at least two amplification reaction chambers, respectively, and having a third depth;

wherein the first depth is greater than the second depth, and the second depth is greater than the third depth.

28. The method of manufacturing according to claim 27, further comprising:

arranging a cover layer above the first side of the body, so as to seal the amplification reaction chambers, the cover layer being provided with at least a first valve hole that extends through the cover layer and overlaps, at least partially, the secondary channels; and

arranging a flexible layer above the cover layer, the flexible layer being provided with at least a protrusion extending through the first valve hole towards the body and overlapping, at least partially, at least one of the at least two secondary channels.

29. A method of manufacturing a microfluidic device, comprising:

forming, at a first side of a body, at least two amplification reaction chambers for storing reagents and samples, an inlet region that is an entrance for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

at least two secondary channels each fluidically connecting the main channel to a respective one of the at least two amplification reaction chambers, respectively, the body also having a second side opposite to the first side;

arranging a cover layer above the first side of the body, so as to seal the amplification reaction chambers, the cover layer having at least a first valve hole that extends through the cover layer and overlaps, at least partially, at least one of the at least two secondary channels; and

arranging a flexible layer above the cover layer, the flexible layer being provided with at least a protrusion extending through the first valve hole towards the body and overlapping, at least partially, at least one of the at least two secondary channels.

31. A method of performing a chemical analysis, said method comprising:

applying a sample to the inlet region of the microfluidic device including a body having a first side and a second side opposite the first side, said first side having;

at least two amplification reaction chambers;

an inlet region forming an entrance for a fluid to be supplied to the amplification reaction chambers;

a main channel fluidically connected to the inlet region; and

at least two secondary channels, each one of the secondary channels fluidically connecting the main channel to a respective one of the amplification reaction chambers, wherein the amplification reaction chambers extend within the body for a first depth, the main channel extends within the body for a second depth, the secondary channels extend within the body for a third depth, the first depth being greater than the second depth, and the second depth being greater than the third depth;

inserting said microfluidic device into a reader device, activating said reader device and obtaining a result of a chemical analysis.

32. The method of claim 31, wherein said chemical analysis is a DNA amplification and detection of same, and said sample is a biological fluid.

33. The method of claim 31, wherein said chemical analysis is an antibody-antigen binding reaction and detection of same, and said sample is a biological fluid.

34. The method of claim 31, wherein said chemical analysis is an enzymatic reaction and detection of same, and said sample is a biological fluid.