COMPOSITIONS AND METHODS FOR DEHYDRATED STORAGE OF ON-BOARD REAGENTS IN MICROFLUIDIC DEVICES

• US 20120156750A1
• Filed: 12/12/2011
• Published: 06/21/2012
• Est. Priority Date: 06/12/2009
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Micronics Inc. (Sony Group Corp.)
• Inventors: Battrell, C. Frederick, Hoekstra, Denise Maxine, Haab, Joan, Williford, John R., Sprague, Isaac, Leber, Elizabeth R., Gates, Sabrina N.
• Granted Patent: US 8,921,085 B2
• Time in Patent Office: Days
• US Class Current: 435/188
• 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 2527/125 Specific component of sample, medium or buffer

Abstract

Manufacturing methods and compositions are described for production of self-contained microfluidic cartridge devices with on-board reagents for molecular biological testing. Sensitive reagents are stored in dry form without lyophilization or freezing, and reconstituted at the point of use with either a biological sample or a sample eluate at the point of use. Manufacturing methods include sheet and roll fabrication processes where the reagents are printed in place and sealed within individual microfluidic cartridges before gel vitrification.

Claims

1. A microfluidic cartridge containing on-board reagents for performing a molecular diagnostic assay, said microfluidic cartridge comprising a plastic body with enclosed microfluidic works having at least one chamber or channel, said chamber or channel having an upstream fluid path joined to a sample inlet port and a downstream fluid path joined to a vent, wherein enclosed within said chamber or channel is at least one vitrified gel reagent spot assembled therein by:

a) combining a first reagent with a printing and stabilizing solution, and printing a droplet of said first printing and stabilizing solution in said chamber or channel and drying said droplet, thereby forming a first gel reagent spot therein;

b) optionally combining a second reagent with a second printing and stabilizing solution, printing a droplet of said second printing and stabilizing solution in said chamber or channel and drying said droplet, thereby forming a second gel reagent spot therein;

c) optionally continuing as in step b until a plurality of on-board gel reagent spots are spotted therein;

d) enclosing said chamber or channel in said plastic body by laminating, gluing, ultrasonically welding, or otherwise attaching a cover or covers thereover; and

e) then sealing said microfluidic cartridge with enclosed gel reagent spot or spots in a gas tight pouch under a dry atmosphere with a dessicant, said dessicant further vitrifying said gel reagent spot or spots during storage, said microfluidic cartridge having an extended shelf life without need of lyophilization or frozen storage and containing all on-board reagents required for a molecular diagnostic assay.

2. The microfluidic cartridge of claim 1, wherein said first vitrified gel reagent spot comprises a TAQ polymerase.

3. The microfluidic cartridge of claim 1, wherein said first vitrified gel reagent spot comprises a TAQ polymerase in a stabilizing matrix comprising:

i) from about 1.0% to 10% w/v of a trisaccharide;

ii) optionally from about 0.001% to 0.1% w/v of a high molecular weight polyethylene glycol or polyhydroxyl compound;

iii) optionally from about 0.001% to 0.3% of a fluorosurfactant;

iv) optionally a non-protein compound having an amino or amide functional group;

v) from about 0.1% to 10% of a carrier protein; and

vi) a compatible buffer.

4. The microfluidic cartridge of claim 1, wherein said second vitrified gel reagent spot comprises a primer or primers.

5. The microfluidic cartridge of claim 1, wherein said plurality of vitrified gel reagent spots comprises a spot containing a probe or probes, a spot containing a reverse transcriptase, a spot containing an RNA polymerase, a spot containing nucleotide triphosphates, a spot containing a nucleic acid template, or a spot containing a buffer.

6. The microfluidic cartridge of claim 1, wherein said microfluidic works comprises a plurality of chambers or channels fluidly connected as a parallel array of assay pathways between said sample inlet port and said vent, wherein within each said assay pathway is at least one vitrified gel reagent spot printed therein.

7. The microfluidic cartridge of claim 6, wherein within each said assay pathway is a vitrified gel reagent spot comprising at least one unique primer pair.

8. The microfluidic cartridge of claim 6, wherein within each said assay pathway is a vitrified gel reagent spot comprising at least one probe.

9. The microfluidic cartridge of claim 6, wherein with each said assay pathway comprises a vitrified gel reagent spot having a TAQ polymerase in a stabilizing matrix.

10. A sheet of microfluidic cartridges of claim 1 or 6 as a process intermediate, said sheet comprises a tessellation of detachable microfluidic cartridges, each said detachable cartridge of said tessellation having been formed with at least one vitrified gel reagent spot assembled therein.

11. The sheet of microfluidic cartridges of claim 10, wherein each said detachable microfluidic cartridge is printed with a pattern of vitrified gel reagent spots configured for a particular molecular diagnostic assay or set of assays.

12. The sheet of microfluidic cartridges of claim 11, wherein each sheet comprises detachable microfluidic cartridges configured for multiple diagnostic assays or sets of assays.

13. The microfluidic cartridge of claim 1 or 6, wherein said at least one vitrified gel reagent spot further comprises a PCR enhancer selected to form a glass at room temperature.

14. The microfluidic cartridge of claim 13, wherein said PCR enhancer is betaine, n-formyl morpholine, δ

-valerolactam (2-piperidone), ∈

-caprolactam, 1,2-cyclopentanediol, PVP-10, PVP-40, or a mixture thereof.

15. The microfluidic cartridge of claim 1 or 6, wherein said at least one vitrified gel reagent spot further comprises inulin, cellulose, derivatized cellulose, polyvinylpyrrolidone, lysine, arginine, or a Maillard reaction inhibitor.

16. A kit comprising a microfluidic cartridge of any of the above claims individually packaged in a gas tight pouch under a dry atmosphere with a dessicant, with labeling and instructions for use.

17. A method for stabilizing a nucleic acid assay reagent for storage in a gel-like glass above the freezing temperature of water without lyophilization in a chamber or channel of a microfluidic device, the method comprising:

a) combining said nucleic acid assay reagent with an aqueous solution composed of;

i) from about 1.0% to 10% w/v of a disaccharide or a trisaccharide;

ii) optionally from about 0.001% to 0.1% w/v of a high molecular weight polyethylene glycol;

iii) optionally from about 0.001% to 0.3% of a fluorosurfactant;

iv) optionally from about 0.001% to about 1.0% of an amino acid;

iv) optionally from about 0.1% to 10% of a carrier protein; and

v) optionally a compatible buffer, thereby forming a composite glass precursor solution;

b) depositing in said microfluidic device a droplet of said composite glass precursor solution containing a quantity of said nucleic acid assay reagent effective for said assay;

c) drying said droplet at controlled room temperature or about 20°

C. degrees to form a gel spot on said surface; and

d) then closing and sealing said gel spot in said microfluidic cartridge in a gas tight pouch under a dry atmosphere with a dessicant, said dessicant further vitrifying said gel spot during storage.

18. The method of claim 17, wherein said disaccharide is selected from trehalose and said trisaccharide is selected from melezitose or raffinose.

19. The method of claim 17, wherein said high molecular weight polyethylene glycol has a molecular weight of 0.1 to 5 MDa and is a linear chain polyoxyethylene glycol or a branched chain polyoxyethylene glycol.

20. The method of claim 17, wherein said carrier protein is bovine serum albumin or fish gelatin.

21. The method of claim 17, wherein said fluorosurfactant is a non-ionic fluoroalkylsurfactant, preferably having a short chain alkyl side chain.

22. The method of claim 17, wherein said fluorosurfactant is Fluorosurfactant FC-4430.

23. The method of claim 17, further comprising a step for passivating said chamber or channel prior to depositing said droplet.

24. The steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps, features, integers, compositions and/or compounds.