Method and materials for isolation of nucleic acid materials

• US 9,382,532 B2
• Filed: 01/30/2014
• Issued: 07/05/2016
• Est. Priority Date: 10/25/2012
• Status: Active Grant

• Current Assignee: Neumodx Molecular, Inc. (Qiagen NV)
• Original Assignee: Neumodx Molecular, Inc. (Qiagen NV)
• Inventors: Brahmasandra, Sundaresh, Mastronardi, Michelle, Craig, Elizabeth, Carey, Maureen
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US14/168,760
• Publication Number:
  US 20140147892A1
• Time in Patent Office: 887 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 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 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• 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 1/701 Specific hybridization probes

Abstract

A method for nucleic acid isolation comprising: receiving a binding moiety solution within a process chamber; mixing the binding moiety solution with a biological sample, within the process chamber, in order to produce a moiety-sample mixture; incubating the moiety-sample mixture during a time window, thereby producing a solution comprising a set of moiety-bound nucleic acid particles and a waste volume; separating the set of moiety-bound nucleic acid particles from the waste volume; washing the set of moiety-bound nucleic acid particles; and releasing a nucleic acid sample from the set of moiety-bound nucleic acid particles. The method preferably utilizes a binding moiety comprising at least one of poly(allylamine) and polypropylenimine tetramine dendrimer, both of which reversibly bind and unbind to nucleic acids based upon environmental pH.

Claims

1. A method for nucleic acid isolation comprising:

receiving a binding moiety solution within a process chamber, wherein the binding moiety solution comprises a collection buffer and a set of affinity moiety-coated microparticles, wherein the set of affinity moiety-coated microparticles comprises Polypropylenimine tetramine dendrimer Generation 1 amide-bonded to a set of microparticles;

contacting the binding moiety solution with a biological sample, within the process chamber, thereby producing a moiety-sample mixture;

incubating the moiety-sample mixture to reversibly bind nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles, thereby producing a set of moiety-bound nucleic acid particles;

receiving a cartridge at a cartridge receiving module comprising a set of pins, each pin in the set of pins displaceable between a first position and a second position;

occluding a fluidic pathway of the cartridge upon displacement of a first subset of the set of pins;

separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture, within the fluidic pathway of the cartridge;

washing the set of moiety-bound nucleic acid particles within the fluidic pathway of the cartridge, in coordination with displacement of a second subset of the set of pins, wherein displacement of the second subset of pins includes reversal of displacement of at least one pin in the first subset of the set of pins, in reversing occlusion of a portion of the fluidic pathway; and

releasing a nucleic acid sample from the set of moiety-bound nucleic acid particles with an elution solution characterized by a pH greater than 10.

2. The method of claim 1, wherein receiving the binding moiety solution, comprising the set of affinity moiety-coated microparticles, within the process chamber comprises receiving a set of magnetic microparticles, each magnetic microparticle modified with an amine-reactive ester functional group configured to react with one of a set of amine groups of a molecule of Polypropylenimine tetramine dendrimer Generation 1.

3. The method of claim 1, wherein receiving the binding moiety solution, comprising the set of affinity moiety-coated microparticles, comprises receiving a set of magnetic microparticles covalently bonded to a set of affinity moiety molecules, and wherein contacting the binding moiety solution with the biological sample comprises aspirating the binding moiety solution along with the biological sample from the process chamber and dispensing the binding moiety solution with the biological sample to the process chamber.

4. The method of claim 3, wherein incubating the moiety-sample mixture comprises heating the moiety-sample solution to a temperature in the range of 25-95°

C. for 5-60 minutes, thus simultaneously lysing the biological sample and facilitating binding of nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles.

5. The method of claim 3, wherein separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture.

6. The method of claim 5, wherein magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises aspirating the moiety-sample mixture from the process chamber;

occluding the fluidic pathway of the cartridge-at a set of occlusion positions upon displacement of pins of the set of pins to generate a path through a magnetic field, and to a waste chamber; and

delivering the moiety-sample mixture through the path of the fluidic pathway, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field and a waste volume of the moiety-sample mixture is delivered into the waste chamber.

7. The method of claim 1, wherein separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises at least one of filtering the moiety-sample mixture and focusing the moiety-sample mixture.

8. The method of claim 1 wherein receiving the binding moiety solution comprises receiving a binding buffer that is also configured to aid in lysis of nucleic acid-containing cells of the biological sample, thereby releasing nucleic acid material from the biological sample for binding.

9. The method of claim 1 wherein receiving the binding moiety solution comprises receiving a binding buffer configured to aid in mitigation of nucleolytic activity from the biological sample.

10. The method of claim 3, wherein washing the set of moiety-bound nucleic acid particles comprises aspirating the moiety-sample mixture;

receiving the cartridge at the cartridge receiving module comprising the set of pins and a cam card interfacing with the set of pins;

transitioning the cartridge to an active configuration that disposes the cartridge proximal the set of pins;

displacing the first subset of the set of pins in the active configuration of the cartridge, upon translation of the cam card relative to the set of pins, thereby occluding the fluidic pathway of the cartridge at a set of occlusion positions;

delivering the moiety-sample mixture into the fluidic pathway, wherein the fluidic pathway is configured to pass through a magnetic field and is coupled to a waste chamber, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field; and

delivering a wash solution through the fluidic pathway and into the waste chamber in a continuous flow operation.

11. The method of claim 1, wherein washing the set of moiety-bound nucleic acid particles comprises transferring the moiety-sample mixture to a fluid vessel, capturing the set of moiety-bound nucleic acid particles within the fluid vessel, and washing the set of moiety-bound nucleic acid particles using a series of aspiration and dispensing steps.

12. The method of claim 11, wherein washing comprises delivering a wash solution through the fluid vessel at a flow rate between 1 and 10 microliters/second, with a total wash solution volume of between 50 and 2000 microliters.

13. The method of claim 3, wherein releasing the nucleic acid sample from the set of moiety-bound nucleic acid particles comprises occluding the fluidic pathway of the cartridge, comprising the process chamber, at a set of occlusion positions, upon displacement of pins of the set of pins, to define a path through a magnetic field;

delivering the set of moiety-bound nucleic acid particles into the path of the fluidic pathway; and

delivering an elution solution into the fluidic pathway, thereby inducing a pH shift configured to release the nucleic acid sample from the set of moiety-bound nucleic acid particles.

14. The method of claim 13, further comprising capturing the set of moiety-bound nucleic acid particles within the magnetic field and aspirating the nucleic acid sample from the fluidic pathway.

15. The method of claim 1, wherein releasing the nucleic acid sample from the set of moiety-bound nucleic acid particles comprises contacting the set of moiety-bound nucleic acid particles with an elution solution characterized by a pH that is in the range of pH 10 to pH 13.

16. The method of claim 1, further comprising processing the nucleic acid sample, wherein processing the nucleic acid sample comprises modulating the fluidic pathway of the cartridge upon displacement of a third subset of the set of pins, thereby defining a path to a diagnostic chamber of the cartridge, delivering the nucleic acid sample to the diagnostic chamber, and amplifying nucleic acids of the nucleic acid sample within the diagnostic chamber by polymerase chain reaction.

17. A method for total nucleic acid extraction, comprising simultaneous isolation and concentration of DNA and RNA from a biological sample, the method comprising:

receiving a binding moiety solution within a process chamber, wherein the binding moiety solution comprises a collection buffer and a set of affinity moiety-coated microparticles, wherein the set of affinity moiety-coated microparticles comprises Polypropylenimine tetramine dendrimer Generation 1 amide-bonded to a set of magnetic microparticles;

mixing the binding moiety solution with between 10uL and 2mL of a biological sample, within the process chamber, thereby producing a moiety-sample mixture;

incubating the moiety-sample mixture to reversibly bind nucleic acid material, comprising target DNA and RNA, of the biological sample to the set of affinity moiety-coated microparticles, thereby producing a set of moiety-bound nucleic acid particles;

receiving a cartridge at a cartridge receiving module comprising a set of pins, each pin in the set of pins displaceable between a first position and a second position;

occluding a fluidic pathway of the cartridge upon displacement of a first subset of the set of pins;

magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture, within the fluidic pathway of the cartridge; and

eluting a nucleic acid sample comprising target DNA and RNA from the set of moiety-bound nucleic acid particles, into less than 20uL of an elution solution characterized by a pH greater than 10, in coordination with displacement of a second subset of the set of pins to modulate flow in the fluidic pathway, thereby facilitating total nucleic acid extraction.

18. The method of claim 17, wherein magnetically separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture comprises aspirating the moiety-sample mixture from the process chamber;

occluding the fluidic pathway of the cartridge at a set of occlusion positions upon displacement of the first subset of the set of pins to generate a path through a magnetic field, and to a waste chamber of the cartridge; and

delivering the moiety-sample mixture through the path of the fluidic pathway, such that the set of moiety-bound nucleic acid particles is captured within the magnetic field and a waste volume of the moiety-sample mixture is delivered into the waste chamber.

19. A method for nucleic acid isolation from a biological sample, comprising:

receiving a binding moiety solution within a process chamber, wherein the binding moiety solution includes a set of affinity moiety-coated microparticles, comprising a set of microparticles amide-bonded to at least one of Poly(allylamine) of molecular weight <

30,000 Da and Polypropylenimine tetramine dendrimer Generation 1;

producing a moiety-sample mixture upon combination of the binding moiety solution with the biological sample;

producing a set of moiety-bound nucleic acid particles upon incubation of the moiety-sample mixture to reversibly bind nucleic acid material of the biological sample to the set of affinity moiety-coated microparticles;

receiving a cartridge at a cartridge receiving module comprising a set of pins, each pin in the set of pins displaceable between a first position and a second position;

occluding a fluidic pathway of the cartridge upon displacement of a first subset of the set of pins;

separating the set of moiety-bound nucleic acid particles from the moiety-sample mixture, within the fluidic pathway of the cartridge;

washing the set of moiety-bound nucleic acid particles within the fluidic pathway of the cartridge; and

releasing a nucleic acid sample from the set of moiety-bound nucleic acid particles with an elution solution, in coordination with displacement of a second subset of the set of pins, wherein displacement of the second subset of the set of pins includes reversing displacement of at least one pin of the first subset of the set of pins, and reversing occlusion of a portion of the fluidic pathway.

20. The method of claim 19, wherein occluding the fluidic pathway comprises linearly displacing a cam card of the cartridge receiving module, comprising a set of hills and valleys, relative to the set of pins, wherein displacing the cam card transitions at least one pin of the set of pins between the first position and the second position.