Processing polynucleotide-containing samples

• US 8,470,586 B2
• Filed: 05/03/2005
• Issued: 06/25/2013
• Est. Priority Date: 05/03/2004
• Status: Active Grant

• Current Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Original Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Inventors: Wu, Betty, Althaus, John S., Brahmasandra, Sundaresh N., Handique, Kalyan, Phadke, Nikhil
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US11/579,353
• Publication Number:
  US 20080262213A1
• Time in Patent Office: 2,975 Days
• Field of Search: None
• US Class Current: 435/283.1
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 2400/0694 vents used to stop and induce flow, backpressure valves
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/5082 : Test tubes per se
• CPC Class Codes: B01L 3/523 : with means for closing or opening
• CPC Class Codes: B01L 3/565 : Seals in general F16L
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12N 15/1006 : by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 : by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0019 : Valves using a microdroplet or microbubble as the valve member
• CPC Class Codes: F16K 99/0032 : using phase transition or influencing viscosity
• CPC Class Codes: F16K 99/0034 : Operating means specially adapted for microvalves
• CPC Class Codes: F16K 99/0036 : operated by temperature variations
• CPC Class Codes: F16K 99/0044 : using thermo-electric means

Abstract

Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that regain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.

Claims

1. A microfluidic device, comprising:

a processing region having a vertical central axis therethrough, the processing region comprised of an inlet, a retention member, a filter, and an outlet;

said retention member being configured to preferentially retain one or more polynucleotides in a sample as compared to polymerase chain reaction inhibitors in the sample, wherein said retention member comprises a plurality of polynucleotide binding particles, and said plurality of binding particles having surfaces that comprise a poly-cationic polyamide bound thereto, further wherein the retention member is configured to retain polynucleotides from a sample preferentially to inhibitors in the sample as the sample passes through the retention member from the processing region inlet to the processing region outlet;

wherein the vertical central axis of the processing region is perpendicular to a horizontal plane of the microfluidic device;

wherein the processing region outlet is disposed along the vertical central axis above the processing region inlet;

wherein the filter is configured to prevent the plurality of binding particles from passing downstream of the processing region;

a device inlet in communication with, and upstream of, the processing region;

a device outlet in communication with, and downstream of, the processing region; and

a detection region in fluid communication with and downstream of the retention member.

2. The microfluidic device of claim 1, wherein the plurality of particles have a volume less than 5 microliters.

3. The microfluidic device of claim 1, wherein the sample has a volume from 0.5 microliters to 3 milliliters.

4. The microfluidic device of claim 1, wherein the poly-cationic polyamide comprises at least one of poly-DL-ornithine, PEI, poly-L-lysine and poly-D-lysine.

5. The microfluidic device of claim 1, wherein the filter is disposed between the retention member and the processing region outlet.

6. The microfluidic device of claim 1, further comprising at least one channel having at least one sub-millimeter cross-sectional dimension.

7. The microfluidic device of claim 1, further comprising a valve, wherein the valve includes a mass of thermally responsive substance that is relatively immobile at a first temperature and more mobile at a second temperature.

8. The microfluidic device of claim 1, further comprising a gate, wherein the gate includes a mass of thermally responsive substance which is relatively immobile at a first temperature and which, upon heating to a second temperature, changes state.

9. The microfluidic device of claim 1, further comprising a fluid reservoir configured to hold a liquid.

10. The microfluidic device of claim 9, wherein the reservoir comprises a liquid having a pH of at least about 10.

11. The microfluidic device of claim 9, wherein the device is configured to contact the retention member with the liquid.

12. The microfluidic device of claim 1, further comprising an actuator, wherein the actuator comprises a chamber having a mass of thermally expansive material therein, and a gas.

13. The microfluidic device of claim 1, further comprising a hydrophobic vent, wherein the vent comprises a layer of porous hydrophobic material.

14. The microfluidic device of claim 1, having three layers of polymeric material.

15. The microfluidic device of claim 1, thermally associated with an array of heat sources.

16. The microfluidic device of claim 15, wherein at least one of the heat sources is configured to heat an aqueous liquid in contact with the retention member to at least about 65°

C.

17. The microfluidic device of claim 16, wherein the liquid is held in a pouch, isolated from the reservoir by a generally impermeable membrane.

18. The microfluidic device of claim 17, wherein the pouch additionally comprises a piercing member configured to puncture the impermeable membrane.

19. The microfluidic device of claim 1, further comprising one or more PCR reagents stored as one or more lyophilized particles.

20. The microfluidic device of claim 19, wherein the lyophilized particles have an average volume of about 5 microliters or less.

21. The microfluidic device of claim 1, further comprising one or more lysing reagents stored as one or more lyophilized particles.

22. The microfluidic device of claim 21, wherein the lyophilized particles have an average volume of about 35 microliters or less.

23. The microfluidic device of claim 1, additionally comprising a lysing chamber and a waste chamber.

24. The microfluidic device of claim 1, wherein the particles are made of a polymeric material selected from the group consisting of:

polystyrene, latex polymers, polyacrylamide, and polyethylene oxide.

25. The microfluidic device of claim 24, wherein the polymeric material is modified to include one or more carboxylic groups and/or one or more amino groups covalently bound to the polymeric material, wherein the groups provide an attachment point for one or more ligands of the poly-cationic polyamide.

26. The microfluidic device of claim 1, wherein the particles have an average diameter of between about 4 microns and about 20 microns.

27. The microfluidic device of claim 1, wherein the particles are present in a density of about 10⁸ particles per milliliter.

28. The microfluidic device of claim 1, wherein at least some of the particles are magnetic.