INTEGRATED NUCLEIC ACID ASSAYS

• US 20120329142A1
• Filed: 06/08/2012
• Published: 12/27/2012
• Est. Priority Date: 03/15/2006
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Micronics Inc. (Sony Group Corp.)
• Inventors: BATTRELL, C. Frederick, GERDES, John, WILLIFORD, John R., HOEKSTRA, Denise Maxine, BREIDFORD, Wayne L., MORDUE, Stephen, CLEMMENS, John, NABAVI, Melud, KOKORIS, Mark
• Granted Patent: US 8,772,017 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/082 Handling hazardous material
• 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 2300/0663 Whole sensors
• 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 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• CPC Class Codes: B01L 2400/0655 pinch valves
• 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: 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 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/131 : the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device

Abstract

Integrated microfluidic cartridges for nucleic acid extraction, amplification, and detection from clinical samples are disclosed. The devices are single-entry, sanitary, and disposable. The devices enable simplex or multiplex nucleic acid target detection, as for example: assay panels for multiple infectious agents, or assay panels for cancerous cell types. Methods for use of microfluidic cartridges in a fully automated, pneumatically controlled apparatus are also disclosed.

Claims

1. A microfluidic cartridge for extracting a nucleic acid fraction from a liquid sample, the microfluidic cartridge comprising:

a) a cartridge body with an inlet port to admit the liquid sample;

b) a lysis chamber having a valved connection with said inlet port,

c) a first fluid reservoir having a valved connection with said lysis chamber, said first fluid reservoir having a lysis reagent contained therein and a pneumatically actuatable diaphragm for dispensing and mixing said lysis reagent into the liquid sample in the lysis chamber, thereby forming a sample lysate;

d) a nucleic acid capture chamber with solid phase adsorbent for capturing the nucleic acid fraction from the sample lysate;

wherein said capture chamber and said lysis chamber are valvedly connected for contacting the sample lysate with the solid phase adsorbent;

e) a second fluid reservoir having a valved connection with said capture chamber, said second fluid reservoir having a wash reagent contained therein and a pneumatically actuatable diaphragm for dispensing the wash reagent so as to wash the solid phase adsorbent layer and discharge any waste sample lysate and washings to a waste outlet in fluid connection with said capture chamber;

f) a third fluid reservoir having a valved connection with said capture chamber, said third fluid reservoir having a nucleic acid elution reagent contained therein and a pneumatically actuatable diaphragm for dispensing the elution reagent onto said solid phase adsorbent so as to elute a nucleic acid eluate; and

,

g) a nucleic acid eluate receiving outlet for receiving the nucleic acid eluate from said capture chamber.

2. The microfluidic cartridge of claim 1, wherein the first fluid reservoir comprises a pneumatic actuator inlet for receiving pneumatic pulses, and further wherein said pneumatic pulses are configured for reciprocatingly actuating said pneumatically actuatable diaphragm of said first reservoir to enable flow of the lysate back and forth between the lysis chamber and the first fluid dispensing chamber so as to enable the reduction of the molecular weight of the nucleic acid fraction.

3. The microfluidic cartridge of claim 1, wherein said pneumatically actuatable diaphragms are configured to be actuated with applied positive pressure strokes and suction pressure strokes.

4. The microfluidic cartridge of claim 3, wherein said applied positive pressure strokes and suction pressure strokes enable reciprocating flow through said valved connections.

5. The microfluidic cartridge of claim 1, wherein the third reservoir comprises a pneumatic actuator inlet for receiving pneumatic pulses configured for actuating said pneumatically actuatable diaphragm to enable slug elution of said nucleic acid eluate.

6. The microfluidic cartridge of claim 4, wherein said applied positive pressure strokes and suction pressure strokes are configured to enable fragmentation of high molecular weight nucleic acid into lower molecular weight fragments.

7. The microfluidic cartridge of claim 1, wherein each of said fluid reservoirs comprise a reagent fluid filled pouch enclosed by a rupturable film, and said pneumatically actuatable diaphragm is configured to pneumatically rupture the pouch.

8. The microfluidic cartridge of claim 1, wherein said lysis reagent comprises a chaeotrope-detergent mixture;

said wash reagent is a nucleic acid precipitant; and

the elution reagent is an aqueous reagent.

9. The microfluidic cartridge of claim 1, wherein said elution reagent is formulated to rehydrate a mixture containing reagents and enzymes.

10. The microfluidic cartridge of claim 1, wherein said elution reagent is formulated for direct use in the amplification of said nucleic acid eluate.

11. The microfluidic cartridge of claim 1, wherein said nucleic acid eluate is extracted for medical or environmental testing.

12. The microfluidic cartridge of claim 1, wherein said lysis chamber is provided with a suction port for aspirating the liquid sample under suction applied thereto, said suction port having a water impermeable, gas permeable membrane.

13. The microfluidic cartridge of claim 12, wherein the water impermeable, gas permeable membrane is hydrophobic.

14. The microfluidic cartridge of claim 12, wherein the water impermeable, gas permeable membrane is hydrophilic.

28. The microfluidic cartridge of claim 12, wherein the elastomeric film covers the absorbent pad.

29. A system comprising the microfluidic cartridge of claim 1 and an off-cartridge pneumatic actuation manifold and apparatus configured with pneumatic interface to said cartridge body, wherein said apparatus is programmed to actuate said pneumatically actuatable diaphragms according to a train of positive and negative pneumatic pressure pulses delivered to said pneumatic interface under control of a microprocessor.

15. A microfluidic cartridge for extracting a nucleic acid fraction from a sample, comprising:

a) a loading port for introduction of the sample;

b) a waste chamber;

c) a lysis sub-circuit operably linked to said loading port, wherein said cell lysis sub-circuit comprises;

     1) a mixing chamber;

     2) a sealed pouch containing lysis reagent; and

     3) a pump member configured for pumping said lysis reagent into said mixing chamber, wherein said lysis sub-circuit is configured to generate a lysed sample from the sample in said mixing chamber; and

d) a nucleic acid extraction sub-circuit operably linked to both of said lysis sub-circuit and said waste chamber, wherein said nucleic acid extraction sub-circuit comprises;

     1) a solid phase nucleic acid extraction component configured to bind nucleic acids present in said lysed sample;

     2) a sealed pouch containing wash reagent;

     3) a sealed pouch containing elution reagent; and

     4) a pump member configured for pumping said elution reagent over said solid phase nucleic acid extraction component to generate a mixture of extracted nucleic acids.

16. The microfluidic cartridge of claim 15, wherein said pump members are pneumatically driven by applied positive pressure strokes and suction pressure strokes.

17. The microfluidic cartridge of claim 16, wherein said applied positive pressure strokes and suction pressure strokes enable reciprocating flow through said valved connections.

18. The microfluidic cartridge of claim 15, wherein said lysis reagent is pumped through a valved connection into said mixing chamber of said lysis subcircuit and said elution reagent is pumped through a valved connection to said solid phase nucleic acid extraction component of said nucleic acid extraction subcircuit.

19. The microfluidic cartridge of claim 17, wherein the pump member for pumping said elution reagent is a pneumatically actuatable diaphragm configured to enable slug elution of said mixture of extracted nucleic acids.

20. The microfluidic cartridge of claim 17, wherein said applied positive pressure strokes and suction pressure strokes are configured to enable fragmentation of high molecular weight nucleic acid into fragments of lower molecular weight.

21. The microfluidic cartridge of claim 15, wherein said lysis reagent comprises a chaeotrope-detergent mixture;

said wash reagent is a nucleic acid precipitant; and

said elution reagent is an aqueous buffer.

22. The microfluidic cartridge of claim 15, wherein said elution reagent is formulated to rehydrate a mixture containing reagents and enzymes.

23. The microfluidic cartridge of claim 15, wherein said nucleic acid eluate is extracted for medical or environmental testing.

24. The microfluidic cartridge of claim 15, wherein said mixing chamber is provided with a suction port for aspirating the liquid sample thereinto, said suction port having a water impermeable, gas permeable membrane.

25. The microfluidic cartridge of claim 24, wherein the water impermeable, gas permeable membrane is hydrophobic.

26. The microfluidic cartridge of claim 24, wherein the water impermeable, gas permeable membrane is hydrophilic.

27. The microfluidic cartridge of claim 15, wherein said waste chamber comprises a sanitary waste collection chamber for capturing waste fluids on the microfluidic cartridge, said waste collection chamber having a fluid inlet and valved fluid connection to said nucleic acid extraction subcircuit, an absorbent pad within said chamber, and an elastomeric or flexible film separating said fluid inlet from an outside vent, said outside vent for venting said waste chamber through said external surface.