INTEGRATED NUCLEIC ACID ASSAYS

• US 20090148933A1
• Filed: 09/10/2008
• Published: 06/11/2009
• Est. Priority Date: 03/15/2006
• Status: Active Grant

• Current Assignee: Perkinelmer Health Sciences Incorporated (Perkinelmer Incorporated)
• Original Assignee: Micronics Inc. (Sony Group Corp.)
• Inventors: Gerdes, John, Hoekstra, Denise Maxine, Clemmens, John, Battrell, C. Frederick, Williford, John R., Breidford, Wayne L., Mordue, Stephen, Kokoris, Mark, Nabavi, Melud
• Granted Patent: US 8,222,023 B2
• Time in Patent Office: Days
• US Class Current: 435/287.200
• 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

1-37. -37. (canceled)

38. An integrated microfluidic cartridge comprising:

(a) a means for extracting nucleic acids from a biosample, comprising a first fluidic subcircuit for extracting nucleic acids from a biosample, said first fluidic subcircuit having a solid phase nucleic acid affinity binding substrate;

(b) a means for synthesizing amplicons, comprising a second fluidic subcircuit for synthesizing amplicons; and

(c) a means for detecting amplicons, comprising a third fluidic subcircuit having a detection chamber and an optical window,

wherein said first, second and third fluidic subcircuits are formed in a single integrated disposable member, and wherein said first, second and third fluidic subcircuits are fluidly interconnected and are controllable by a plurality of internal pneumatic control elements comprising diaphragm-operated valves and bellows chambers, and wherein said plurality of internal pneumatic control elements are provided with pneumatic interconnect circuitry for connection to an external controllable pneumatic actuation manifold.

39. The microfluidic cartridge of claim 38, wherein said means for synthesizing amplicons further comprises:

(a) a first bellows chamber fluidly interconnected with a second bellows chamber; and

(b) a thermal interface, whereby the first and second bellows chambers are held at different fixed temperatures,

wherein each bellows chamber further comprises a diaphragm for reciprocal pumping of fluid between the first and second bellows chambers.

41. A method of performing PCR in the microfluidic cartridge of claim 39 comprising:

(a) forming a reaction mixture comprising a DNA template, primers, polymerase, dNTPs, buffer and a magnesium salt in said first bellows chamber, said first bellows chamber being held at a fixed temperature at or above the denaturation temperature of double stranded DNA and said second bellows chamber being held at a fixed temperature at or below the annealing temperature of said DNA template and said primers;

(b) introducing a nucleic acid target sequence into said first bellows chamber;

(c) applying pneumatic pressure to said diaphragm of said first bellows chamber so as to effect transfer of said reaction mixture from said first bellows chamber to said second bellows chamber;

(d) applying pneumatic pressure to said diaphragm of said second bellows chamber so as to effect transfer of said reaction mixture from said second bellows chamber to said first bellows chamber;

(e) repeating steps (c) and (d) for a number of thermocycles so as to synthesize amplification products; and

(f) removing the amplification products.

40. (canceled)

42-54. -54. (canceled)

55. An apparatus for performing a nucleic acid assay on a biological sample, said apparatus comprising:

(a) a plastic disposable cartridge body containing dry and fluid reagents for said nucleic acid assay, wherein said cartridge body comprises;

(i) a first fluidic subcircuit for extracting a nucleic acid from said sample;

(ii) a second fluidic subcircuit for synthesizing an amplicon product;

(iii) a third fluidic subcircuit for detecting said amplicon product;

(iv) a sanitary waste collection chamber; and

(v) a pneumatic interconnect subcircuit with arms for pneumatically actuating said first, second and third fluidic subcircuits; and

(b) an off-cartridge pneumatic actuation manifold with arms and solenoid valves for supplying a programmable train of positive and negative pneumatic pressure pulses to said pneumatic interconnect subcircuit of said cartridge body under control of a microprocessor configured for controlling said nucleic acid assay.

56. The apparatus of claim 55, wherein said first fluidic subcircuit comprises:

(a) a sample inlet port;

(b) a first bellows pump fluidly and valvedly interconnected by a first fluidic channel to said sample inlet port and by a second fluidic channel to a second bellows pump, wherein said second bellows pump comprises a frangible lysis buffer reagent pack for programmable admixture of lysis buffer to said sample and wherein said pneumatic interconnect subcircuit is configured for programmable rupture of said lysis buffer reagent pack by compressing said lysis buffer reagent pack between a diaphragm and an opposing sharp in said second bellows pump; and

(c) a solid-phase nucleic acid target capture assembly for extracting a nucleic acid, wherein said target capture assembly is fluidly and valvedly interconnected to said first bellows pump.

57. The apparatus of claim 56, wherein said first fluidic subcircuit further comprises:

(a) a third bellows pump fluidly and valvedly interconnected to said target capture assembly, wherein said third bellows pump comprises a frangible wash reagent pack for programmable delivery of a wash reagent to said target capture assembly and wherein said pneumatic interconnect subcircuit is configured for programmable rupture of said wash reagent pack by compressing said wash reagent pack between a diaphragm and an opposing sharp in said third bellows pump; and

(b) a sanitary waste receptacle fluidly and valvedly connected by a waste fluidic channel to said target capture assembly.

58. The apparatus of claim 57, wherein said first fluidic subcircuit further comprises:

(a) a fourth bellows pump fluidly and valvedly interconnected to said target capture assembly, wherein said fourth bellows pump comprises a frangible elution reagent pack for programmable delivery of an elution buffer to said target capture assembly and wherein said pneumatic interconnect subcircuit is configured for programmable rupture of said elution reagent pack by compressing said elution reagent pack between a diaphragm and an opposing sharp in said fourth bellows pump; and

(b) a second fluidic channel for interconnecting said target capture assembly with said second fluidic subcircuit, whereby a nucleic acid eluate is conveyed to said second fluidic subcircuit for amplification.

59. The apparatus of claim 58, wherein said second fluidic subcircuit comprises:

(a) a PCR fluidics and thermal interface comprising a first and second bellows pump fluidly interconnected by a microchannel, wherein said PCR fluidics and thermal interface is configured for holding said first bellows pump at a fixed temperature for denaturing said nucleic acid eluate and said second bellows pump at a fixed temperature for annealing said nucleic acid eluate, and wherein said programmable train of positive and negative pneumatic pressure pulses is configured for reciprocally pumping of said nucleic acid eluate between said first and second bellows pumps, thereby thermocycling said nucleic acid eluate;

(b) a dehydrated PCR mix reagent with primers for reconstitution in said nucleic eluate; and

(c) a third fluidic channel with valve for valvedly interconnecting said second fluidic circuit with said third fluidic subcircuit, whereby said amplicon product of said second fluidic circuit is conveyed to said third fluidic subcircuit for detection.

60. The apparatus of claim 59, wherein said dehydrated PCR mix reagent is directly reconstituted in said nucleic acid eluate.

61. The apparatus of claim 59, wherein said first and second bellows pumps of said PCR fluidics and thermal interface are held at said fixed temperatures by heating and cooling means, said heating and cooling means comprising first and second heating and cooling blocks contacting said thermal interface, wherein said first block operatively contacts said first bellows pump and said second block operatively contacts said second bellows pump.

62. The apparatus of claim 59, wherein said second bellows pump comprises a dehydrated polymerase.

63. The apparatus of claim 61, further comprising a cDNA synthesis chamber fluidically interposed between said target capture assembly and said first and second bellows pumps of said PCR fluidics and thermal interface.

64. The apparatus of claim 61, wherein said sanitary waste receptacle comprises a fluid inlet for waste, a bibulous pad within said receptacle, and a film covering said bibulous pad and separating said fluid inlet from an outside vent for venting said waste receptacle.