Integrated nucleic acid assays

• US 8,772,017 B2
• Filed: 06/08/2012
• Issued: 07/08/2014
• 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
• Primary Examiner(s): Beisner, William H
• Application Number:
  US13/492,612
• Publication Number:
  US 20120329142A1
• Time in Patent Office: 760 Days
• Field of Search: 435/288.5
• 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 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;

wherein said microfluidic cartridge is configured to pump said lysis reagent through a valved connection into said mixing chamber of said lysis subcircuit and said microfluidic cartridge is configured to pump said elution reagent through a valved connection to said solid phase nucleic acid extraction component of said nucleic acid extraction subcircuit;

wherein said pump members are configured to be pneumatically driven by applied positive pressure strokes and suction pressure strokes;

wherein said applied positive pressure strokes and suction pressure strokes enable reciprocating flow through said valved connections; and

wherein the pump member for pumping said elution reagent is a pneumatically actuatable, elastomeric diaphragm configured to enable slug elution of said mixture of extracted nucleic acids.

2. The microfluidic cartridge of claim 1, 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.

3. The microfluidic cartridge of claim 1, 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.

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

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

6. The microfluidic cartridge of claim 1, 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.

7. The microfluidic cartridge of claim 6, wherein the water impermeable, gas permeable membrane is hydrophobic.

8. The microfluidic cartridge of claim 6, wherein the water impermeable, gas permeable membrane is hydrophilic.

9. The microfluidic cartridge of claim 1, 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.

10. The microfluidic cartridge of claim 9, wherein the elastomeric film covers the absorbent pad.