Rehydratable matrices for dry storage of TAQ polymerase in a microfluidic device
View Patent ↗Formulations for dry storage of PCR reagents are described. These formulations find use in manufacture of self-contained microfluidic card devices for PCR clinical testing in which the reagents are reconstituted at the point of testing. In these cards, TAQ polymerase is stored “on-board” in vitrified dry form without lyophilization or freezing, and is reconstituted by either the sample or a sample eluate during the assay.
1. A method for stabilizing a TAQ polymerase for printing and storage in a microfluidic cartridge, the method comprising:
a) combining said TAQ polymerase with an aqueous solution to form a printable solution, the aqueous solution 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 (PEG);
iii) from about 0.001% to 0.3% w/v of a fluorinated surfactant;
iv) from about 0.1 mg/ml to 10 mg/ml of a carrier protein; and
v) a compatible buffer;
b) depositing on a surface of the microfluidic cartridge a droplet of said printable solution containing a quantity of said TAQ polymerase effective in polymerizing a nucleic acid;
c) drying said droplet at a controlled room temperature to form a gel spot on said surface; and
d) closing and sealing said gel spot on said surface in a gas tight pouch under a dry atmosphere with a dessicant, said dessicant further vitrifying said gel spot during storage.
2. The method of claim 1 , wherein said trisaccharide is melezitose or raffinose.
3. The method of claim 1 , wherein said high molecular weight polyethylene glycol is PEG90M.
4. The method of claim 1 , wherein said high molecular weight polyethylene glycol has a molecular weight of 1 to 5 MDa.
5. The method of claim 1 , wherein said carrier protein is bovine serum albumin or fish gelatin.
6. The method of claim 1 , wherein said fluorinated surfactant is a non-ionic fluoroalkylsurfactant.
7. The method of claim 6 , wherein said fluorinated surfactant is Fluorosurfactant FC-4430.
8. The method of claim 1 , wherein said aqueous solution further comprises a PCR enhancer selected from betaine, n-formyl morpholine, δ-valerolactam (2-piperidone), ε-caprolactam, 1,2-cyclopentanediol, polyvinylpyrrolidone, or a mixture thereof.
9. The method of claim 1 , wherein said aqueous solution further comprises inulin, cellulose, derivatized cellulose, polyvinylpyrrolidone, lysine, arginine, or a Maillard reaction inhibitor.
10. The method of claim 1 , wherein the controlled room temperature is about 20° C. degrees.
11. A method for stabilizing a TAQ polymerase for printing and storage on a microfluidic cartridge, without lyophilization, said method comprising:
a) combining said TAQ polymerase with an aqueous solution to form a solution, said aqueous solution 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 (PEG);
iii) from about 0.001% to 0.3% of a fluorinated surfactant;
iv) from about 0.1 mg/ml to 10 mg/ml of a carrier protein; and
v) a compatible buffer;
b) depositing on a surface of the microfluidic cartridge a droplet of said printable TAQ solution comprising a quantity of said TAQ polymerase effective for polymerizing a nucleic acid;
c) drying said droplet at a controlled room temperature to form a partially vitrified gel spot on said surface; and
d) closing and sealing said gel spot on said surface in a gas tight pouch under a dry atmosphere with a dessicant, said dessicant further vitrifying said gel spot during storage.
12. The method of claim 11 , wherein said trisaccharide is melezitose or raffinose.
13. The method of claim 11 , wherein said high molecular weight polyethylene glycol is PEG90M.
14. The method of claim 11 , wherein said high molecular weight polyethylene glycol has a molecular weight of 1 to 5 MDa.
15. The method of claim 11 , wherein said carrier protein is bovine serum albumin or fish gelatin.
16. The method of claim 11 , wherein said fluorinated surfactant is a non-ionic fluoroalkylsurfactant.
17. The method of claim 16 , wherein said fluorinated surfactant is Fluorosurfactant FC-4430.
18. The method of claim 11 , wherein said aqueous solution further comprises a PCR enhancer selected from betaine, n-formyl morpholine, δ-valerolactam (2-piperidone), ε-caprolactam, 1,2-cyclopentanediol, polyvinylpyrrolidone, or a mixture thereof.
19. The method of claim 11 , wherein said aqueous solution further comprises inulin, cellulose, derivatized cellulose, polyvinylpyrrolidone, lysine, arginine, or a Maillard reaction inhibitor.
20. The method of claim 11 , wherein the controlled room temperature is about 20° C. degrees.