Compositions on plasma-treated surfaces

Abstract

To provide solid compositions such as lyophilizates adhering to surfaces such as plasma-treated surfaces, and related methods, uses, kits, intermediates, starting materials and downstream products.SOLUTION: A method of preparing a solid composition adhering to a plasma-treated surface comprises drying a solution on the plasma-treated surface, and forming a solid composition from the solution, the solid composition adhering to the plasma-treated surface. In some embodiments, the plasma-treated surface is a surface treated with a cold cathode discharge, hollow cathode discharge, DC-induced discharge, radio frequency (RF)-induced discharge, corona discharge, glow discharge, or charged particle beam.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a sequel to Provisional Application No. 62 / 402,446, filed September 30, 2016. The benefit of priority under 35 U.S.C. §119(e) to the application Ser. No. 119 / 002,313, filed on Dec. 13, 2002, the entire contents of which are incorporated by reference. No. 6,399,433, filed on Oct. 23, 2003, and is incorporated herein by reference in its entirety. Summary of the Invention

[0002] Solid compositions do not necessarily adhere strongly to surfaces. Lack of sufficient adhesion can result in chemical Solid pharmaceutical compositions for carrying out biological, pharmaceutical, or biochemical reactions or assays or In fields such as providing reagents, the composition may shift during shipping or handling, or The problem is that the dislocated or loosened composition may become loose. The reconstitution liquid is dispensed into the device and some or all of the composition is no longer at the bottom of the reservoir where reconstitution occurs. Failure to attach to or near the site of the fusion protein may complicate or prevent reconstitution. The inability to completely reconstitute the composition to a liquid state can adversely affect assay performance. It's possible.

[0003] For example, filler reagents or reagents for carrying out chemical or biochemical reactions or assays. The composition including the reagent is provided as a solid composition attached to the plasma-treated surface. The plasma treatment is carried out by applying a solid composition formed by drying a liquid solution to a surface. and that the plasma treatment can improve adhesion to surfaces at or near the bottom of the vessel. Prevent or reduce the frequency of dislocation or loosening of the solid composition from a surface such as Without wishing to be bound by any particular theory, it has been found that plasma treatment The treatment can increase the hydrophilicity of the surface and therefore its wettability, enhancing the interaction of the reagent with the surface. The enhanced interaction persists even after a drying process, such as freeze-drying.

[0004] The present disclosure relates to a method for adhering a solid composition, such as a freeze-dried pellet, to a surface, such as a plastic, by: The insight that the solid composition can be strengthened by plasma treatment of the surface before contacting the surface. Thus, the compositions, methods, and kits disclosed herein are based in part on: Improved ability to survive agitation that may occur during processes such as shipping and handling We can provide products with the above capabilities.

[0005] Accordingly, disclosed herein is a method for preparing a solid composition attached to a plasma-treated surface. The method includes drying the solution on the plasma treated surface and forming a solid composition from the solution. and forming a solid composition on the plasma-treated surface, the solid composition adhering to the plasma-treated surface.

[0006] In some embodiments, the plasma treated surface may be a cold cathode discharge, a hollow cathode discharge, a DC induction Treated with electrical discharge, radio frequency (RF) inductive discharge, corona discharge, glow discharge, or charged particle beam In some embodiments, the plasma treated surface is a corona discharge treated surface. In some embodiments, the method comprises about 25 Watts / minute / m 2 ~ approx. 2000 watts / min / m 2 , about 50 watts / min / m 2 ~ approx. 1500 watts / min / m 2 , about 100 to about 12 00 watts / min / m 2 , about 200 to about 1000 watts / min / m 2 , about 100 to about 600 t / min / m 2, or about 200 watts / min / m 2 ~ approx. 600 watts / min / m 2 In the range of Plasma-treated surfaces were prepared by treating the surface with a corona discharge at a watt density. In some embodiments, the surface is heated to about 25 Watts / min / m 2 ~about 20 00 watts / min / m 2 , about 50 watts / min / m 2 ~ approx. 1500 watts / min / m 2 , about 10 0 to approx. 1200 watts / min / m 2 , about 200 to about 1000 watts / min / m 2 , about 100~ Approximately 600 watts / min / m 2 , or about 200 watts / min / m 2 ~ approx. 600 watts / min / m 2 In some embodiments, the electrodes were treated with a corona discharge at a watt density in the range of 1000 to 15000 Hz. i) The plasma treated surface is at about 33 to about 45 dynes, about 35 to about 42 dynes, or about 3 having a surface energy in the range of 7 to about 40 dynes, and optionally the surface comprises a polyolefin. or (ii) the plasma treated surface is at about 35 to about 55 dynes, about 37 to about 53 dynes. or a surface energy in the range of about 40 to about 50 dynes, and optionally the surface is In some embodiments, the plasma treated surface comprises polyethylene or polypropylene. has an increase in surface energy compared to an untreated surface of the same composition, and the increase in surface energy The heating is performed at about 3 to about 20 dynes, about 4 to about 15 dynes, about 5 to about 12 dynes, or about 6 to about It is in the 10 dyne range.

[0007] In some embodiments, drying can be achieved by evaporation, dehydration, desiccating, freezing, or the like. In some embodiments, the method includes at least one of freeze-drying, sublimation, and spray-drying. In some embodiments, the solution is frozen during at least a portion of the drying step. Drying includes freeze-drying. In some embodiments, the solution is at least one of the drying steps. In some embodiments, the drying occurs under vacuum. In embodiments, drying occurs at a temperature greater than about 30° C. In some embodiments, the solution Before drying, about 5 μL to about 20 mL, about 200 μL to about 20 mL, about 1 mL to about 20 mL L, about 5 mL to about 20 mL, about 5 μL to about 1 mL, about 5 μL to about 500 μL, about 5 μL to Approximately 200μL, approximately 5μL to approximately 100μL, approximately 5μL to approximately 50μL, approximately 5μL to approximately 20μL , about 5 μL to about 10 μL, about 10 μL to about 200 μL, about 20 μL to about 200 μL, about 5 The volume ranges from about 0 μL to about 200 μL, or from about 100 μL to about 200 μL.

[0008] In some embodiments, the plasma treatment surface is an interior surface of a plasma treatment vessel. In some embodiments, the plasma treated surface comprises plastic. So, plastics are polyethylene, polyethylene terephthalate, polypropylene, Polymethacrylate, polyvinyl chloride, polystyrene, polyolefin, polycarbonate Polyurethane, starch-derived plastics, or cyclic olefin copolymers In some embodiments, the plastic comprises at least one of a cyclic olefin copolymer. Includes polymers.

[0009] In some embodiments, the solution comprises water. In some embodiments, the solution comprises a polar The polar organic solvent is ethanol, isopropanol, DMSO, or 21. The method of claim 20, wherein the at least one of the following is selected from the group consisting of glycerol and glycerol. In an embodiment, the solution comprises a non-polar organic solvent.

[0010] In some embodiments, the solution comprises a bulking agent. In some embodiments, the bulking agent In some embodiments, the bulking agent is sucrose, mannitol, glycerin, or the like. At least one of hydroxyethyl starch, raffinose, or trehalose Alternatively, the bulking agent may be sucrose, mannitol, raffinose, or and trehalose.

[0011] In some embodiments, the solution comprises one or more enzymes. One or more of the enzymes may be a DNA polymerase, an RNA polymerase, a ligase, a kinase, or a phosphatase. Among the sphatases, proteases, exonucleases, and endonucleases In some embodiments, the one or more enzymes comprise at least one thermostable DNA polymerase. In some embodiments, the thermostable DNA polymerase comprises a hot-stable DNA polymerase. Some are thermostable DNA polymerases or conjugated to antibodies. In embodiments, the one or more enzymes are present at a concentration of about 0.20 U / uL to about 0.72 U / uL or about 0 A DNA-dependent polymerase present in the solution at a concentration of about 0.1 U / uL to about 0.6 U / uL. The enzyme may include a ribozyme, a reverse transcriptase, or other enzymes.

[0012] In some embodiments, the solution comprises a buffer. In some embodiments, the buffer , an organic buffering agent.

[0013] In some embodiments, the solution has an inorganic salt concentration of about 5 mM or less. In embodiments, the solution does not include inorganic salts. In some embodiments, the solution includes salts.

[0014] In some embodiments, the solution comprises at least one ointment useful for performing a molecular assay. In some embodiments, at least one oligonucleotide In some embodiments, the oligode is selected from an amplification oligomer and a probe. The nucleotide comprises a label. In some embodiments, the label is fluorescent, chemiluminescent, affinity or radioactively labeled.

[0015] In some embodiments, the solution comprises one or more nucleoside triphosphates. In some embodiments, the solution includes one or more deoxynucleoside triphosphates. In an embodiment, the solution contains dATP, dGTP, dCTP, and dTTP and dUT In some embodiments, one or more nucleoside trinucleotides are At least one of the phosphates is about 0.1 mM to about 0.3 mM or about 0.2 mM in the solution. In some embodiments, the solution is present in an amount ranging from about 0.5 mM to about 0.6 mM. The probe comprises at least one probe selected from a probe and a hydrolysis probe.

[0016] In some embodiments, the solution comprises an RNase inhibitor. The RNase inhibitor is present in the solution at a concentration of about 0.12 U / μL to about 0.20 U / μL. do.

[0017] In some embodiments, the solution includes a chelating agent. The chelating agent is EDTA, EDDS, or MGDA. The dosing agent is present in the solution at a concentration of about 1.5 mM to about 2.0 mM.

[0018] In some embodiments, the solution comprises a detergent. In some embodiments, the detergent is a non- The detergent may be ionic, cationic, anionic, or zwitterionic.

[0019] In some embodiments, the solution contains a single unit dose of amplification reagent. In embodiments, the solution contains a single unit dose of detection reagent. The liquid contains a pharma- ceutically active agent.

[0020] In some embodiments, the method further comprises applying a solution onto the plasma-treated surface prior to the drying step. In some embodiments, the method further comprises pre-treating the surface prior to the dispensing step. In some embodiments, the method further comprises: The method includes sealing the solid composition in a vessel containing a plasma-treated surface after the drying step. nothing.

[0021] Cyclic olefin containing liquid or frozen solution on the concave surface of a cyclic olefin copolymer surface A copolymer surface is also provided, the cyclic olefin copolymer surface having a viscosity of about 35 dynes to about 55 dynes. The liquid or frozen solution has a surface energy or contact angle of about 5° to about 50°. Contains sugar.

[0022] In some embodiments, the liquid or freezing solution comprises water. In some embodiments, the liquid or frozen solution comprises a polar organic solvent. is at least one of ethanol, isopropanol, DMSO, or glycerol. In some embodiments, the liquid or frozen solution comprises a non-polar organic solvent.

[0023] In some embodiments, the liquid or frozen solution comprises one or more enzymes. In embodiments, the one or more enzymes are a DNA polymerase, an RNA polymerase, a ligase , kinases, phosphatases, proteases, exonucleases, and endonucleases The protein comprises at least one of the following:

[0024] In some embodiments, the liquid or freezing solution comprises a buffer. In this case, the liquid or frozen solution is free of inorganic salts or has an inorganic salt concentration of about 5 mM or less. In some embodiments, the liquid or freezing solution comprises a salt.

[0025] In some embodiments, the liquid or frozen solution is useful for performing molecular assays. In some embodiments, the nucleic acid comprises at least one oligonucleotide. The solution includes one or more nucleoside triphosphates.

[0026] In some embodiments, the solution is a liquid. In some embodiments, the solution is a frozen It is tied.

[0027] Also provided is a solid composition adhered to a plasma-treated cyclic olefin copolymer surface, The solid composition is within a concave surface of the cyclic olefin copolymer surface, the concave surface being at an angle of about 90 degrees to about 60 degrees. 0 degree wall angle, and the solid composition has a mass of about 600 micrograms to about 100 mg. The cyclic olefin copolymer containing the solid composition has a surface area of ​​up to about 480 mm After package performance testing in which the solid composition is dropped from a height of 100 mm onto a stainless steel surface, The particles are sufficiently adherent to the crystalline olefin copolymer surface to remain substantially adhered thereto. A solid composition attached to the plasma-treated surface is also provided, The composition is produced by the methods disclosed herein. A solid composition is also provided, the solid composition comprising a filler and one of the enzyme and oligonucleotide. Includes at least one and.

[0028] In some embodiments, the plasma treated surface comprises plastic. In terms of form, plastics are polyethylene, polyethylene terephthalate, polypropylene, Polymethacrylate, polyvinyl chloride, polystyrene, polyolefin, polycarbonate esters, polyurethanes, starch-derived plastics, or cyclic olefin copolymers In some embodiments, the plastic comprises at least one of cyclic olefins. Copolymers.

[0029] In some embodiments, the filler is a sugar, glycine, or hydroxyethyl starch. In some embodiments, the sugars include sucrose, mannitol, raffinose. In some embodiments, the solid composition comprises at least one of: The composition is in a chamber that includes a plasma-treated surface.

[0030] In some embodiments, the reservoir comprises a tube having a volume ranging from about 10 μL to about 60 mL. In some embodiments, the vessel is a multi-well plate containing multiple wells.

[0031] In some embodiments, the solid composition contains from about 5 mg to about 20 g, from about 200 mg to about 20 g, about 1g to about 20g, about 5g to about 20g, about 5mg to about 1g, about 5mg to about 500mg , about 5mg to about 200mg, about 5mg to about 100mg, about 5mg to about 50mg, about 5mg ~about 20mg, about 5mg to about 10mg, about 10mg to about 200mg, about 20mg to about 20 0 mg, about 50 mg to about 200 mg, or about 100 mg to about 200 mg Yes.

[0032] In some embodiments, the solid composition comprises one or more enzymes. In the case of ribozymes, one or more of the enzymes are DNA polymerase, RNA polymerase, ligase, kinase, etc. of enzymes, phosphatases, proteases, exonucleases, and endonucleases Including at least one of them.

[0033] In some embodiments, the solid composition comprises a buffer. In some embodiments, the solid composition is free of inorganic salts or is or has an inorganic salt concentration of about 5 mM or less.

[0034] In some embodiments, the solid composition comprises at least one molecule useful for performing a molecular assay. In some embodiments, the solid composition comprises one or more oligonucleotides. In some embodiments, the solid composition comprises a single unit dose of a nucleoside triphosphate. In some embodiments, the solid composition contains an amplification or detection reagent for a pharma- ceutical active agent. Contains harmful drugs.

[0035] Kits are also provided that include a reconstitution liquid and a solid composition disclosed herein. Also provided is a method of preparing a reconstituted liquid and solid composition as disclosed herein. by combining to form a reconstituted composition.

[0036] In some embodiments, the reconstitution liquid is a mixture of water, a polar organic solvent, and a non-polar organic solvent. In some embodiments, the reconstitution liquid comprises water. In some embodiments, the reconstitution liquid comprises at least one inorganic salt.

[0037] In some embodiments, the methods disclosed herein further comprise adding a nucleic acid. wherein the nucleic acid is subjected to at least one nucleic acid modification or hybridization reaction. In some embodiments, the nucleic acid modification or hybridization reaction is a nucleic acid synthesis or In some embodiments, the nucleic acid modification or hybridization reaction includes amplification. hybridizing the probe to a nucleic acid to form a hybridized complex. .

[0038] In some embodiments, the methods disclosed herein comprise hybridizing the probe. The method further comprises detecting the cytoplasmic complex or nuclear lysis.

[0039] The section headings are provided for the convenience of the reader and are not intended to limit this disclosure. should not be interpreted as In certain embodiments, for example, the following are provided: (Item 1) 1. A method for preparing a solid composition adhered to a plasma-treated surface, the method comprising drying a solution on the plasma-treated surface and forming a solid composition from the solution, wherein the solid composition adheres to the plasma-treated surface. (Item 2) 2. The method of claim 1, wherein the plasma-treated surface is a surface treated with cold cathode discharge, hollow cathode discharge, DC inductive discharge, radio frequency (RF) inductive discharge, corona discharge, glow discharge, or a charged particle beam. (Item 3) The method according to claim 1 or 2, wherein the plasma-treated surface is a corona discharge-treated surface. The method comprises: 2 ~ approx. 2000 watts / min / m 2 , about 50 watts / min / m 2 ~ approx. 1500 watts / min / m 2 , about 100 to about 1200 watts / min / m 2 , about 200 to about 1000 watts / min / m 2 , about 100 to about 600 watts / min / m 2 , or about 200 watts / min / m 2 ~ approx. 600 watts / min / m 2 4. The method of claim 3, comprising preparing the plasma-treated surface by treating the surface with a corona discharge at a watt density in the range of (Item 5) The surface is about 25 watts / min / m 2 ~ approx. 2000 watts / min / m 2 , about 50 watts / min / m 2 ~ approx. 1500 watts / min / m 2 , about 100 to about 1200 watts / min / m 2 , about 200 to about 1000 watts / min / m 2 , about 100 to about 600 watts / min / m 2 , or about 200 watts / min / m 2 ~ approx. 600 watts / min / m 2 4. The method of claim 3, wherein the substrate is treated with a corona discharge at a watt density in the range of 0.1 to 1.0 watts. (Item 6) 6. The method according to any one of items 1 to 5, wherein (i) the plasma-treated surface has a surface energy in the range of about 33 to about 45 dynes, about 35 to about 42 dynes, or about 37 to about 40 dynes, and optionally the surface comprises a polyolefin; or (ii) the plasma-treated surface has a surface energy in the range of about 35 to about 55 dynes, about 37 to about 53 dynes, or about 40 to about 50 dynes, and optionally the surface comprises polyethylene or polypropylene. (Item 7) 7. The method according to any one of items 1 to 6, wherein the plasma-treated surface has an increase in surface energy compared to an untreated surface of the same composition, and the increase in surface energy is in the range of about 3 to about 20 dynes, about 4 to about 15 dynes, about 5 to about 12 dynes, or about 6 to about 10 dynes. (Item 8) 8. The method according to any one of items 1 to 7, wherein the drying comprises at least one of evaporation, dehydration, desiccating, freeze-drying, sublimation, and spray-drying. (Item 9) 9. The method according to any one of items 1 to 8, wherein the solution is frozen during at least a portion of the drying step. (Item 10) 10. The method according to any one of items 1 to 9, wherein the drying comprises freeze-drying. (Item 11) 11. The method according to any one of items 1 to 10, wherein the solution is liquid during at least a portion of the drying step. (Item 12) 12. The method according to any one of items 1 to 11, wherein the drying occurs under vacuum. (Item 13) 13. The method according to any one of items 1 to 12, wherein the drying occurs at a temperature above about 30°C. (Item 14) 14. The method according to any one of items 1 to 13, wherein the solution has a volume before drying in the range of about 5 μL to about 20 mL, about 200 μL to about 20 mL, about 1 mL to about 20 mL, about 5 mL to about 20 mL, about 5 μL to about 1 mL, about 5 μL to about 500 μL, about 5 μL to about 200 μL, about 5 μL to about 100 μL, about 5 μL to about 50 μL, about 5 μL to about 20 μL, about 5 μL to about 10 μL, about 10 μL to about 200 μL, about 20 μL to about 200 μL, about 50 μL to about 200 μL, or about 100 μL to about 200 μL. (Item 15) 15. The method according to any one of items 1 to 14, wherein the plasma treatment surface is an inner surface of a plasma treatment vessel. (Item 16) 16. The method of any one of items 1 to 15, wherein the plasma-treated surface comprises plastic. (Item 17) 17. The method of claim 16, wherein the plastic comprises at least one of polyethylene, polyethylene terephthalate, polypropylene, polymethacrylate, polyvinyl chloride, polystyrene, polyolefin, polycarbonate, polyurethane, starch-derived plastic, or cyclic olefin copolymer. (Item 18) 18. The method of claim 17, wherein the plastic comprises a cyclic olefin copolymer. (Item 19) 19. The method according to any one of items 1 to 18, wherein the solution comprises water. (Item 20) 20. The method according to any one of items 1 to 19, wherein the solution comprises a polar organic solvent. (Item 21) 21. The method of claim 20, wherein the polar organic solvent comprises at least one of ethanol, isopropanol, DMSO, or glycerol. (Item 22) 22. The method according to any one of items 1 to 21, wherein the solution comprises a non-polar organic solvent. (Item 23) 23. The method according to any one of items 1 to 22, wherein the solution comprises a filler. (Item 24) 24. The method of claim 23, wherein the filler comprises a sugar. (Item 25) 25. The method according to claim 23 or 24, wherein the filler comprises at least one of sucrose, mannitol, glycine, hydroxyethyl starch, raffinose, or trehalose, or alternatively, the filler is a sugar selected from sucrose, mannitol, raffinose, or trehalose. (Item 26) 26. The method according to any one of items 1 to 25, wherein the solution comprises one or more enzymes. (Item 27) 27. The method of claim 26, wherein the one or more enzymes comprise at least one of a DNA polymerase, an RNA polymerase, a ligase, a kinase, a phosphatase, a protease, an exonuclease, and an endonuclease. (Item 28) 29. The method of claim 27, wherein the one or more enzymes include a thermostable DNA polymerase. 29. The method of claim 28, wherein the thermostable DNA polymerase is a hot-start thermostable DNA polymerase or is conjugated to an antibody. (Item 30) 28. The method of claim 26 or 27, wherein the one or more enzymes comprise a DNA-dependent polymerase, reverse transcriptase, or other enzyme present in the solution at a concentration of about 0.20 U / uL to about 0.72 U / uL or about 0.1 U / uL to about 0.6 U / uL. (Item 31) 31. The method according to any one of items 1 to 30, wherein the solution comprises a buffer. (Item 32) 32. The method of claim 31, wherein the buffer is an organic buffer. (Item 33) 33. The method according to any one of items 1 to 32, wherein the solution has an inorganic salt concentration of about 5 mM or less. (Item 34) 34. The method according to any one of items 1 to 33, wherein the solution does not contain inorganic salts. (Item 35) 35. The method according to any one of items 1 to 34, wherein the solution comprises a salt. (Item 36) 36. The method according to any one of items 1 to 35, wherein the solution comprises at least one oligonucleotide useful for performing a molecular assay. (Item 37) 37. The method of claim 36, wherein the at least one oligonucleotide is selected from an amplification oligomer and a probe. (Item 38) 38. The method of claim 36 or 37, wherein the oligonucleotide comprises a label. (Item 39) 39. The method of claim 38, wherein the label is a fluorescent, chemiluminescent, affinity, or radioactive label. (Item 40) 40. The method according to any one of items 1 to 39, wherein the solution comprises one or more nucleoside triphosphates. (Item 41) 41. The method of claim 40, wherein the solution comprises one or more deoxynucleoside triphosphates. (Item 42) 42. The method of claim 41, wherein the solution comprises at least one of dATP, dGTP, dCTP, and dTTP and dUTP. (Item 43) 43. The method according to any one of items 40 to 42, wherein at least one of the one or more nucleoside triphosphates is present in the solution in an amount ranging from about 0.1 mM to about 0.3 mM or from about 0.2 mM to about 0.6 mM. (Item 44) 44. The method according to any one of items 1 to 43, wherein the solution comprises at least one probe selected from a capture probe and a hydrolysis probe. (Item 45) 45. The method according to any one of items 1 to 44, wherein the solution comprises an RNase inhibitor. (Item 46) Item 46. The method of item 45, wherein the RNase inhibitor is present in the solution at a concentration of about 0.12 U / μL to about 0.20 U / μL. (Item 47) 47. The method according to any one of items 1 to 46, wherein the solution comprises a chelating agent. (Item 48) 48. The method of claim 47, wherein the chelating agent is EDTA, EDDS, or MGDA. (Item 49) 49. The method according to claim 47 or 48, wherein the chelating agent is present in the solution at a concentration of about 1.5 mM to about 2.0 mM. (Item 50) 50. The method according to any one of items 1 to 49, wherein the solution comprises a detergent. (Item 51) 51. The method of claim 50, wherein the detergent is a nonionic, cationic, anionic, or zwitterionic detergent. (Item 52) 52. The method according to any one of items 1 to 51, wherein the solution contains a single unit dose of amplification reagent. (Item 53) 53. The method according to any one of items 1 to 52, wherein the solution contains a single unit dose of detection reagent. (Item 54) 54. The method according to any one of items 1 to 53, wherein the solution contains a pharma- ceutically active agent. (Item 55) 55. The method according to any one of the preceding claims, wherein the method comprises dispensing the solution onto the plasma-treated surface prior to the drying step. (Item 56) 56. The method of claim 55, wherein the method comprises plasma treating a surface prior to the dispensing step to form the plasma-treated surface. (Item 57) 57. The method according to any one of the preceding claims, further comprising after the drying step sealing the solid composition in a chamber containing the plasma-treated surface. (Item 58) A cyclic olefin copolymer surface containing a liquid or frozen solution on a concave surface of the cyclic olefin copolymer surface, said cyclic olefin copolymer surface having a surface energy of about 35 dynes to about 55 dynes or a contact angle of about 5° to about 50°, and said liquid or frozen solution containing a sugar. (Item 59) 59. The surface of item 58, wherein the liquid or frozen solution comprises water. (Item 60) 61. The surface of claim 58 or 59, wherein the liquid or frozen solution comprises a polar organic solvent. 61. The surface of item 60, wherein the polar organic solvent comprises at least one of ethanol, isopropanol, DMSO, or glycerol. (Item 62) 62. The surface according to any one of items 58 to 61, wherein the liquid or frozen solution comprises a non-polar organic solvent. (Item 63) 63. The surface according to any one of items 58 to 62, wherein the liquid or frozen solution comprises one or more enzymes. (Item 64) 64. The surface of item 63, wherein the one or more enzymes comprise at least one of a DNA polymerase, an RNA polymerase, a ligase, a kinase, a phosphatase, a protease, an exonuclease, and an endonuclease. (Item 65) 65. The surface according to any one of items 58 to 64, wherein the liquid or freezing solution comprises a buffer. (Item 66) 66. The surface according to any one of items 58 to 65, wherein the liquid or freezing solution is free of inorganic salts or has an inorganic salt concentration of about 5 mM or less. (Item 67) Item 68. The surface according to any one of items 58 to 66, wherein the liquid or freezing solution comprises a salt. 68. The surface according to any one of items 58 to 67, wherein the liquid or frozen solution comprises at least one oligonucleotide useful for performing a molecular assay. (Item 69) 69. The surface according to any one of items 58 to 68, wherein the liquid or frozen solution comprises one or more nucleoside triphosphates. (Item 70) 70. The surface according to any one of items 58 to 69, wherein the solution is a liquid. (Item 71) 70. The surface according to any one of items 58 to 69, wherein the solution is frozen. (Item 72) 1. A solid composition adhered to a plasma-treated cyclic olefin copolymer surface, said solid composition being within a concave surface of said cyclic olefin copolymer surface, said concave surface having a wall angle of from about 90 degrees to about 60 degrees, said solid composition having a mass of from about 600 micrograms to about 100 mg, and comprising a sugar, said solid composition being sufficiently adherent such that said solid composition remains substantially adhered to said cyclic olefin copolymer surface after a packaging performance test in which said cyclic olefin copolymer surface containing said solid composition is dropped from a height of up to about 480 mm onto a stainless steel surface. (Item 73) 54. A solid composition adhered to a plasma-treated surface, wherein the solid composition adhered to the plasma-treated surface is produced by the method according to any one of items 1 to 53. (Item 74) A solid composition attached to a plasma-treated surface, said solid composition comprising a filler and at least one of an enzyme and an oligonucleotide. (Item 75) 75. The solid composition according to claim 73 or 74, wherein the plasma-treated surface comprises a plastic. (Item 76) 76. The solid composition according to claim 75, wherein the plastic comprises at least one of polyethylene, polyethylene terephthalate, polypropylene, polymethacrylate, polyvinyl chloride, polystyrene, polyolefin, polycarbonate, polyurethane, starch-derived plastic, or cyclic olefin copolymer. (Item 77) 77. The solid composition according to claim 75 or 76, wherein the plastic comprises a cyclic olefin copolymer. (Item 78) 78. The solid composition according to any one of items 73 to 77, wherein the filler comprises a sugar, glycine, or hydroxyethyl starch. (Item 79) 79. The solid composition according to item 72 or 78, wherein the sugar comprises at least one of sucrose, mannitol, raffinose, or trehalose. (Item 80) 80. The solid composition according to any one of items 72 to 79, wherein the solid composition is in a vessel comprising the plasma-treated surface. (Item 81) Item 81. The solid composition according to item 80, wherein the reservoir comprises a tube having a volume ranging from about 40 μL to about 60 mL. (Item 82) 81. The solid composition according to item 80, wherein the vessel is a multiwell plate containing a plurality of wells. (Item 83) 83. The solid composition according to any one of items 72 to 82, wherein the solid composition has a mass in the range of about 5 mg to about 20 g, about 200 mg to about 20 g, about 1 g to about 20 g, about 5 g to about 20 g, about 5 mg to about 1 g, about 5 mg to about 500 mg, about 5 mg to about 200 mg, about 5 mg to about 100 mg, about 5 mg to about 50 mg, about 5 mg to about 20 mg, about 5 mg to about 10 mg, about 10 mg to about 200 mg, about 20 mg to about 200 mg, about 50 mg to about 200 mg, or about 100 mg to about 200 mg. (Item 84) 84. The solid composition according to any one of items 72 to 83, wherein the solid composition comprises one or more enzymes. (Item 85) 85. The solid composition of claim 84, wherein the one or more enzymes comprise at least one of a DNA polymerase, an RNA polymerase, a ligase, a kinase, a phosphatase, a protease, an exonuclease, and an endonuclease. (Item 86) 86. The solid composition according to any one of items 72 to 85, wherein the solid composition comprises a buffer. (Item 87) 87. The solid composition according to any one of items 72 to 86, wherein the solid composition comprises a salt. (Item 88) 88. The solid composition according to any one of items 72 to 87, wherein the solid composition is free of inorganic salts or has an inorganic salt concentration of about 5 mM or less. (Item 89) 89. The solid composition according to any one of items 72 to 88, wherein the solid composition comprises at least one oligonucleotide useful for performing a molecular assay. (Item 90) 90. The solid composition according to any one of items 72 to 89, wherein the solid composition comprises one or more nucleoside triphosphates. (Item 91) 91. The solid composition according to any one of items 72 to 90, wherein the solid composition contains a single unit dose of an amplification or detection reagent. (Item 92) 92. The solid composition according to any one of items 72 to 91, wherein the solid composition contains a pharma- ceutically active agent. (Item 93) A kit comprising a reconstitution liquid and a solid composition according to any one of items 72 to 92. (Item 94) 93. A method for forming a mixture, comprising combining a reconstituted liquid and a solid composition according to any one of items 72 to 92 to form a reconstituted composition. (Item 95) 95. The kit or method of item 93 or 94, wherein the reconstitution liquid comprises at least one of water, a polar organic solvent, and a non-polar organic solvent. (Item 96) 96. The kit or method of item 95, wherein the reconstitution liquid comprises water. (Item 97) 97. The kit or method according to any one of items 93 to 96, wherein the reconstitution liquid comprises at least one inorganic salt. (Item 98) 98. The method according to any one of items 94 to 97, further comprising adding a nucleic acid, wherein said nucleic acid undergoes at least one nucleic acid modification or hybridization reaction. (Item 99) 99. The method of claim 98, wherein the nucleic acid modification or hybridization reaction comprises nucleic acid synthesis or amplification. (Item 100) 100. The method of claim 99, wherein the nucleic acid modification or hybridization reaction comprises hybridizing a probe to the nucleic acid to form a hybridized complex. (Item 101) 101. The method of claim 100, wherein the method further comprises detecting a hybridized complex of the probe or nuclear lysis. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 is a diagram of the contact angle measurement, where θ is the contact angle, H is the height of the droplet, and R is half the width of the droplet. [Figure 2A] 2A-2B show the drop test results of lyophilized pellets in untreated and plasma-treated wells as described in Example 1. The height of the bars indicates the number of drops before the attachment was broken. [Figure 2B] 2A-2B show the drop test results of lyophilized pellets in untreated and plasma-treated wells as described in Example 1. The height of the bars indicates the number of drops before the attachment was broken. [Figure 3A] 3A-3B show the drop test results of lyophilized pellets in untreated and plasma-treated wells as described in Example 2. The height of the bars indicates the number of drops before the attachment was broken. [Figure 3B] 3A-3B show the drop test results of lyophilized pellets in untreated and plasma-treated wells as described in Example 2. The height of the bars indicates the number of drops before the attachment was broken. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] definition Before describing the present teachings in detail, it is important to note that the disclosure is not limited to particular compositions or process steps. It is understood that the present invention is not limited to the above and may vary. When used, the singular forms "a", "an" and "the" shall be used unless the context clearly indicates otherwise. Please note that plural references are included unless otherwise indicated. Thus, for example, , reference to "a filler" includes a plurality of fillers, and so forth.

[0042] Temperatures, concentrations, times, etc. discussed in this disclosure are preceded by an implied "about" which makes It will be understood that minor and insubstantial deviations are within the scope of the teachings herein. In general, the term "about" refers to a range of concentrations that do not significantly affect the activity or stability of the composition. Also, "comprise", "contains" and "contains "comprising", "containing" ain," "contains," "containin g), "include", "includes", and "contains The use of "including" is not intended to be limiting. Both the detailed description and the detailed description are exemplary and explanatory only and are not intended to be limiting teachings. I want you to understand that.

[0043] Unless otherwise stated, embodiments herein recite "comprising" various components. is also contemplated as "consisting of" or "consisting essentially of" the recited components, and various Any embodiment herein that recites "consisting of" an element may include the recited element. "comprising" or "consisting essentially of" are also contemplated, and "essentially consisting of" various components. Any embodiment herein that recites "consisting of" or "consisting of" the recited elements. "comprises" is also contemplated (this interchangeability applies to the use of these terms in the claims). will not be displayed).

[0044] As used herein, the surface is agitated, e.g., shaken (e.g., about 1 to 20 min.). When the material is visibly exposed to the surface (by hand at a frequency of 3 Hz) or inverted, If the material does not move, it is considered to be "adhered" to the surface.

[0045] The "filler" acts as a matrix for the deposition of proteins and other reagents during drying and storage. (Carpenter et al. (2002) Rational data sign of stable lyophilized protein form lations.Kluwer Academic / Plenum, New York, pp.109-133). Fillers are used to give the product a "cake" or other structure. to prevent proteins or other reagents from being lost from the bath during drying. This can increase the stability of the protein.

[0046] Chelating agents are used to remove Mg, which may be required for the activity of certain enzymes. 2+ or Mn 2+ etc. It is a drug that sequesters divalent ions.

[0047] A "concave surface" is a surface that is suitable for holding a solution or a solid composition formed from a solution. The recess, well, depression, or other feature of the surface of the cavity may be a recess, well, depression, or other feature of the surface of the cavity.

[0048] The "contact angle" is the distance between the surface of the substrate and a tangent drawn at a three-phase point to the surface of the droplet on the substrate. See Figure 1. The contact angle θ is given by the formula θ=2(arc tangent (H / R)). where H is the height of the droplet and R is half the width of the droplet. Unless otherwise indicated, the droplet is water for contact angles referred to herein. Contact angle measurements are ASTM International Standard D 5946-04,“S Standard Test Method for Corona-Treated P olymer Films Using Water Contact Angle M The contact angle measurement method and See also U.S. Patent No. 5,268,733 for a discussion of the device. Unless otherwise specified, contact angle measurements are performed using a water drop. When a surface is in contact with water, the contact angle is observed to be at a certain value or at a certain It is considered that the contact angle is in a certain range.

[0049] As used herein, "first," "second," etc., refer to the ordering or position of elements. used to refer to an element of structure, composition, or method without any implication as to placement, Thus, unless one of ordinary skill in the art understands otherwise from the context, a "first" element is a "second" element. It may be before, overlap, after, above, below, before, at the same time, or after the element.

[0050] The terms "lyophilization," "lyophilized," and "freeze-dried" refer to a drying The material to be frozen is first frozen, and then the ice or frozen solvent is evaporated by sublimation in a vacuum environment. "Lyophile" refers to a material that has been freeze-dried.

[0051] The term "stringent" ("stringency") in relation to nucleic acid hybridization The term "optimal hybridization conditions" or "stringent conditions" refers to specific of oligonucleotide hybridizes to the target nucleic acid more than to other nucleic acids present in the test sample. These conditions refer to conditions under which it is possible to the length of the hybridization sequence, the hybridization temperature, the composition of the hybridization reagent or solution, as well as the degree of hybridization specificity desired. It will be understood that suitable hybridization conditions are determined by the nature of the probe, amplification oligonucleotide, and the like. Oligonucleotides, target capture oligonucleotides, blockers, and other oligonucleotides These are well known in the art and can be predicted based on sequence composition or This can be determined by using routine testing methods (see, for example, Sambrook et al. al.,Molecular Cloning,A Laboratory Manual l,2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) §§1.90 -1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 , in particular §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and and 11.55-11.57).

[0052] Amplification oligomers are primers or promoters capable of supporting template-dependent replication. The amplification oligomer pair supports template-dependent replication of opposite strands of the template. Multiplex amplification is the simultaneous use of multiple amplification oligomer pairs. This is an amplification carried out by

[0053] The probe hybridizes to the amplification product and reveals the presence or amount of the amplification product. Such probes are often fluorescent or In some cases, the markers incorporate other detectable signal-providing molecules, in which case they are detectably labeled. These are called recognized probes.

[0054] The primer-probe set is adapted to generate and detect an amplification product from a template nucleic acid. It is a combination of constructed primers and probes.

[0055] "Reconstitution time" refers to the time required to rehydrate the dry formulation with a solution until it is free of particles or turbidity to the naked eye. The time required to obtain a clear solution is

[0056] A "single unit dose" or "SUD" is a dose that is administered to perform an assay on a single sample. Refers to the amount of reagent used. A single unit dose may be a solution. The dosage is a dry pellet containing reagents useful for the amplification of a single sample in a single vessel. Good too.

[0057] As used herein, "or" is used inclusively, i.e., "A or Unless otherwise clearly indicated to the contrary, in expressions such as "one or less of B" and "and is equivalent to "and / or."

[0058] As used herein, "or a combination thereof" means "at least one of The terms "one" and "one or more of" refer to all of the terms in the list that precedes the term. For example, "A, B, C, or any combination of these" At least one of A, B, C, AB, AC, BC, or ABC, in any order. BA, CA, CB, ACB, CBA, BCA, BAC, if important in the given context , or CAB. Continuing with this example, BB, AAA, AAB, BB One or more items or terms such as C, AAABCCCC, CBBAAA, CABABB Combinations involving repetition are also included. Typically, any combination is used unless otherwise clear from the context. Those skilled in the art will understand that there is no limit to the number of items or terms in a combination of .

[0059] "Package performance test" refers to a test in which a surface (e.g., a bath) to which a solid composition is adhered is dropped from a certain height (e.g., about 480 mm) onto a substrate (e.g., stainless steel) to determine the degree of adhesion of the solid composition to the surface. A solid composition is considered to remain substantially adherent if no visible portion of the solid composition becomes loose or if less than about 5% or about 10% by volume becomes loose (e.g., moves relative to the surface when the surface is shaken or inverted). In some embodiments, the height used in the package performance test is about 20 mm to 1000 mm, for example, about 20 mm, about 40 mm, about 60 mm, about 80 mm, about 100 mm, about 120 mm, about 140 mm, about 160 mm, about 180 mm, about 200 mm, about 220 mm, about 240 mm, about 260 mm, about 280 mm, about 300 mm, about 320 mm, about 340 mm, about 360 mm, about 380 mm, about 400 mm, about 420 mm, about 440 mm, about 460 mm, about 480 mm, about 500 mm, about 520 mm, about 540 mm, about 560 mm, about 580 mm, about 600 mm, about 620 mm, about 640 mm, about 660 mm, about 680 mm, about 700 mm, about 720 mm, about 740 mm, about 760 mm, about 780 mm, about 800 mm, about 820 mm, about 840 mm, about 860 mm, about 880 mm, about 900 mm, about 920 mm, about 940 mm, about 960 mm, about 980 mm, or about 1000 mm, or about 20 mm to about 60 mm, about 60 mm to about 100 mm, about 100 mm to about 200 mm, about 200 mm to about 300 mm, about 300 mm to about 400 mm, about 500 mm to about 600 mm, about 600 mm to about 700 mm, about 800 mm to about 900 mm, or about 900 mm to about 1000 mm. In some embodiments, the substrate is a flexible foam, e.g., polyurethane, such as a polyurethane sheet. In some embodiments, the substrate has a resistance of about 18 to about 20 lbs / in 2 In some embodiments, the substrate is a hard surface such as stainless steel. In some embodiments, the substrate has a thickness of about 1 cm, 2 cm, 3 cm, 4 cm, or 5 cm.

[0060] "Plasma" is defined as any mixture of positively and negatively charged particles that is not bound together and is not contained in a container. Plasma is a state of matter that does not have a definite shape or definite volume. It may also contain uncharged particles that are gases in the absence of electrons, e.g., air, O2, N2, etc. Plasma can be produced by applying an electric field to a gas, as in a corona discharge. Other methods of producing plasma include heating a gas, applying electromagnetic radiation (e.g., radio frequency (R F) inductive discharge), and generating charged particle beams, such as electron beams.

[0061] The surface is exposed to the plasma and at least one property affected by the exposure, e.g. , a decrease in contact angle, an increase in surface energy, or a solid composition compared to an untreated counterpart. A surface is "plasma treated" if it has increased adhesion to metals, etc.

[0062] "Sugars" have a carbon to oxygen ratio of approximately (but not necessarily exactly) 1:1. The ratio of hydrogen to oxygen is about 2:1 (but not necessarily exactly) It is a compound that contains hydrogen and oxygen. 12 O 5), as well as glucose (CH 12 O6) and sucrose (C 12 H 22 O 11 ) and other sugars.

[0063] As used herein, a "solid composition" refers to a non-flowable material. Unless otherwise specified, a solid composition is generally a coating such as a dry paint that is thin in one dimension compared to the other. For solid compositions, the process does not mean leaving a non-coated residue. Solid compositions include freeze-dried products, pellets, etc., resulting from drying a solution with ethanol. Although solid compositions are generally crystalline or have a lattice structure at the molecular level, It also includes amorphous and quasicrystalline solids. A solid composition is a solid at 20°C and 1 atm pressure. In some embodiments, the solid composition includes three orthogonal freezing solutions. are macroscopic in some dimensions (i.e., they are observable without the aid of a microscope, e.g. For example, the solid compositions herein have a length, width, and height of greater than about 1 mm. 0.5 to 2 g / cm 3 and their volumes are estimated accordingly. It can be done.

[0064] As used herein, a "solution" is a composition that includes a solvent and a solute dissolved therein. and not just in liquid solutions, e.g., solutes generally remain dispersed in frozen solvents. It also includes frozen solutions, which are the result of freezing a solvent containing dissolved solutes (such as from a solvent). (as opposed to precipitating or otherwise forming a significantly different solid phase).

[0065] Exemplary Methods, Compositions, and Kits In some embodiments, the solution is allowed to dry on the plasma-treated surface, and the plasma-treated surface is A method for preparing an adherent solid composition is provided that forms a solid composition adherent to a surface. In some embodiments, the plasma treated surface is treated with a cold cathode discharge, a hollow cathode discharge, a DC induction Treated with electrical discharge, radio frequency (RF) inductive discharge, corona discharge, glow discharge, or charged particle beam In some embodiments, the plasma-treated surface is a surface that has been treated with a corona discharge. It is a surface that has been

[0066] In some embodiments, a solid composition is provided that is attached to the plasma-treated surface. In some embodiments, the ring includes at least one concave surface having a wall angle of about 90 degrees to about 60 degrees. A cyclic olefin copolymer surface is provided, the cyclic olefin copolymer surface being about 600 mm thick. The solid composition has a mass of about 100 mg to about 100 mg, and contains a solid composition containing a sugar. is attached to the cyclic olefin copolymer surface, and the solid composition is a cyclic olefin copolymer containing solid composition. An olefin copolymer surface is dropped onto a stainless steel surface from a height of approximately 480 mm. After packaging performance testing, it remains substantially attached to the cyclic olefin copolymer surface. In some embodiments, a solid composition is provided that is attached to the plasma-treated surface, the solid composition The article includes a filler and at least one of an enzyme and an oligonucleotide. In some embodiments, the plasma treated surfaces produced by the methods disclosed herein An adherent solid composition is provided.

[0067] The present invention also provides a method for treating a plasma-treated surface comprising administering to the patient a solid composition as disclosed herein, such as a solid composition attached to a plasma-treated surface. Also provided is a kit comprising the composition, optionally further comprising a reconstitution liquid.

[0068] A method of forming a mixture is also provided, the method comprising: By combining the solid compositions disclosed herein, such as a solid composition attached to to form a reconstituted composition.

[0069] In some embodiments, a liquid or frozen solution is applied to the concave surface of the cyclic olefin copolymer surface. A cyclic olefin copolymer surface is provided that contains a cyclic olefin copolymer. The contact angle was decreased or increased compared to the untreated cyclic olefin copolymer. In some embodiments, the cyclic olefin copolymer has a surface energy A cyclic olefin copolymer surface containing a liquid or frozen solution is provided on the concave surface of the cyclic olefin copolymer surface. The olefin copolymer has a surface energy of about 35 dynes to about 55 dynes or about 5 The contact angle is about 50° to 60°.

[0070] The following paragraphs, the Summary of the Invention, the Figures and their Description, the Examples, and the Claims (which are the subject matter of this application) The present application is hereby incorporated by reference in its entirety, without regard to any part of the present application, which is hereby deemed to be part of the specification, or any other part of this application. The details of certain embodiments described herein may be adapted to the extent that they can be applied according to the understanding of those skilled in the art. All of the methods, compositions, and kits disclosed in the book are described herein.

[0071] Treating the surface with plasma can result in the formation of a solid composition on the surface as the solid composition forms during drying of the solution. It is believed that this enhances the extent to which the surface favorably interacts with the molecules of the solid composition. The polymer process allows for non-covalent interactions such as dipole-dipole and hydrogen bonding with the molecules of the solid composition. The charge density or density of partially charged atoms or groups on the surface may be increased by: Thus, a range of plasma treatment durations and intensities, e.g., about 25 watts / min. / m 2 ~ approx. 2000 watts / min / m 2 , about 50 watts / min / m 2 ~ approx. 1500 watts / minute / m 2 , about 100 to about 1200 watts / min / m 2 , about 200 to about 1000 watts / min / m 2, about 100 to about 600 watts / min / m 2 , or about 200 watts / min / m 2 ~about 600 watts / min / m 2 Treatment with plasma sources such as corona discharges at watt densities in the range of It can be used.

[0072] The extent of plasma treatment can also be expressed in terms of surface energy or change in surface energy. In some embodiments, plasma treated surfaces, such as surfaces comprising polyolefins. is in the range of about 33 to about 45 dynes, about 35 to about 42 dynes, or about 37 to about 40 dynes In some embodiments, the polyethylene or polypropylene Plasma treated surfaces, such as surfaces containing fluorine, are typically treated at about 35 to about 55 dynes, about 37 to about 53 dynes or in the range of about 40 to about 50 dynes. showed that plasma-treated surfaces have an increased surface energy compared to untreated surfaces of the same composition. The increase in surface energy is about 3 to about 20 dynes, about 4 to about 15 dynes, and about 5 to about 12 dynes. dynes, or in the range of about 6 to about 10 dynes.

[0073] The extent of plasma treatment can also be expressed in terms of contact angle or change in contact angle. In some embodiments, the plasma treated surface, such as a surface comprising a polyolefin, is heated to about 5° C. to about 5° C. 0°, for example, about 5° to about 40°, about 5° to about 30°, about 5° to about 10°, about 10° to about 20°, approx. 10°~approx. 30°, approx. 10°~approx. 40°, approx. 20°~approx. 30°, approx. 30°~approx. In some embodiments, the contact angle is 40°, or between about 40° and about 50°. In some embodiments, the contact angle is from about 10° to about 25°. In some embodiments, the contact angle is compared to the contact angle of the surface before plasma treatment. The amount of decrease is, in some embodiments, about 30° to about 95°, for example, about 40°. ~90°, ~30°~40°, ~40°~50°, ~50°~60°, ~60° ~70°, ~80°, ~90°, or ~95° The range value is:

[0074] In some embodiments, the plasma treated surface is a plasma treated vessel, e.g., a tube, or In some embodiments, the inner surface of a multi-well plate or cartridge. In some embodiments, the surface or bath comprises plastic. polyethylene, polyethylene terephthalate, polypropylene, polymethacrylate, Vinyl chloride, polystyrene, polyolefin, polycarbonate, polyurethane, starch The plastics used in the present invention include at least one of the following: polyethylene-derived plastic, polyethylene-derived plastic, or cyclic olefin copolymer.

[0075] Apparatus for plasma processing, a method for using such an apparatus, or a method for plasma processing General considerations are found, for example, in WO2002 / 023960(A1), US2009 / 0143 718(A1), US6,106,653, US7,556,845(B2), US3, 870,610, US2013 / 0017672(A1), US8,361,565(B 2) and US9,005,188(B2). It is believed that no plasma treatment was applied in relation to the adhesion of body components. Regardless, given the guidance of this disclosure and the available knowledge regarding plasma processing in general, Thus, those of ordinary skill in the art are fully able to practice, make, and use the compositions and methods disclosed herein. It is possible.

[0076] In some embodiments, drying comprises lyophilization. The composition is a lyophilisate, such as a lyophilised pellet. In some embodiments, the drying is At least one of the following methods may be used: evaporation, dehydration, desiccating, freeze-drying, and spray-drying. In some embodiments, drying is performed under vacuum, e.g., at about 0.9, 0. 8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0 Pressures below .01 atmosphere or approximately 0.9, 0.8, 0.7, 0.6, 0.5, or 1.0 atmospheres above ambient pressure Occurs under pressures of 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 times lower In some embodiments, drying is performed at elevated temperatures, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 5° C. higher, or higher than about 30° C., for example, about 30° C. to 110° C., about 30° C. ℃~40℃, about 40℃~50℃, about 50℃~60℃, about 60℃~70℃, about 70℃~80 ℃, about 80℃~90℃, about 90℃~100℃, or about 100℃~110℃ In some embodiments, drying is performed at ambient temperature, or at about 15° C. to 30° C., at about 15° C. ~25℃, approx. 20℃~30℃, approx. 15℃~20℃, approx. 20℃~25℃, or approx. 25℃ In some embodiments, drying occurs at a temperature of about 100° C. to about 30° C. In some embodiments, drying occurs at a lower temperature, e.g., below ambient temperature. or less than about 1, 2, 3, 4, or 5°C, or less than 15°C, for example, about 10°C to about 15℃, about 5℃ to about 10℃, about 0℃ to about 5℃, about -5℃ to about 0℃, about -10℃ to about 0℃, Approximately -20℃ to approximately -10℃, approximately -30℃ to approximately -20℃, approximately -40℃ to approximately -30℃, approximately -50 ℃ to about -40℃, about -60℃ to about -50℃, about -70℃ to about -60℃, about -80℃ to about - It occurs at temperatures of 70°C, about -90°C to about -80°C, or about -100°C to about -90°C.

[0077] In some embodiments, the solution is frozen during at least a portion of the drying step. In some embodiments, the solution is liquid during at least a portion of the drying step. In some embodiments, the solution is frozen during all or substantially all of the drying step. In some embodiments, the solution remains liquid during all or substantially all of the drying step. In some embodiments, the solution is liquid during part of the drying step and is dissolved. A portion of the solvent undergoes a liquid-to-gas transition, for example during evaporation, and then freezes, and the remaining When a solvent undergoes a solid-to-gas transition, such as in sublimation, during freeze-drying, , is solid during at least a portion of the drying step.

[0078] The solution may be lyophilized using standard methods and equipment. For example, from GEA Process Engineering, Columbia, MD Contract freeze-drying services are available, for example, from Biopharmaceutical Company chnology Ltd.,Winchester,Hampshire,Great Britain and BioPharma Solutions Sterile C ontract Manufacturing,Baxter Healthcare Corp, Deerfield, Ill. Guidance for lyophilization is available at For example, L. Rey, J.C. May (eds.) (2010) Freeze Dry ng / Lyophilization of Pharmaceuticals and Biological Products,3 rd .ed.Informa Heal thcare, NY or Methods in Enzymology, Vol. 2 2,Pages 33-39,Academic Press,New York (1 971), or Freeze-Drying, E. W. Flosdorf, Rhein Hold, New York (1949). Optionally, freeze-dried During the synthesis, the oxygen content can be reduced (Phillips et al. (2001) Biol. ics.19:219).

[0079] A variety of containers are suitable for drying. When the containers are sealed and stored under partial vacuum, The container must be capable of withstanding external pressure. The container must be able to withstand reasonable pressure from the outside to the inside. The size of the container should preferably be such that it is The solution to be added should occupy less than 20% of the total volume to avoid overflowing. It is.

[0080] The samples may be dried in separate chambers or in a multi-analyte chamber. A multi-analyte chamber is one that contains at least two samples. so that they can be stored and operated in parallel, but separately. For multiple specimen vessels, e.g., multi-well plates or cartridges, Standard formats for this are 6, 24, 96, 384 or 1536 concave surfaces, e.g. The volume of each well in an example of a 96-well format is The volume is about 300-400 microliters, and the working volume is about 75-200 microliters. Other sizes are contemplated, but the volume is generally inverse to the number of wells, typically varies from 1 nL to 10 mL per well. Exemplary wells and recesses are: It may have a flat bottom, a round bottom, or a V-shaped bottom, among others. The well may further be referred to as a reaction well. The term reaction well refers to any reaction It is not required that the reaction actually occur within the reaction well. Rather, the term includes , in which there is no response, in which there is a partial response, or in which there is a complete Used to refer to a vessel or well that may contain a reaction.

[0081] As mentioned above, the multi-well plate may be freeze-dried in some embodiments herein. The solution can be subjected to drying to form a solid composition. (See co-pending International Application No. PCT / US2016 / 045166. The nest is closed by a mechanism that can be operated from outside the sealed freeze-drying chamber. A container for plates with vents that can be inserted into the nest containing multi-well plates. is then placed in the freeze-drying chamber with one or more vents in an open position. The chamber is then sealed and a freeze-drying atmosphere is applied to the entire chamber, including the space within the nest. One or more vents are then closed, thereby sealing the nest. The seal on the chamber is later released and the nest containing the multiwell plate is removed. The operator then decides whether to use or further store the lyophilized composition located therein. The multiwell plates containing the lyophilized specimens are ready to be resealed for storage or sale. The nest can then be repositioned and reassembled with the multiwell plate positioned within it. The wells of the multi-well plate are then sealed to protect against moisture from the surrounding air. In some embodiments, the sealed multi-wheat flour can be used to substantially prevent the ingress of The well plates are stored in pouches containing desiccant. Similarly, separate chambers are subjected to lyophilization. The container can be sealed and freeze-dried in the nest. This can prevent small amounts of moisture from entering the multi-well plate.

[0082] Optimal lyophilization conditions are product dependent. In some embodiments, the lyophilization procedure comprises: For example, the heat treatment step may include a heat treatment step at a temperature of about -30°C to about -10°C. The heat treatment step may have a duration of, for example, 30 minutes to 6 hours. For example, the heat treatment step may have a duration of about -2 0° C. for about 2 hours. In some embodiments, the freeze-drying procedure may be performed at, e.g., For example, the drying step may include a primary drying step at a temperature of about -50°C to about -10°C. The loop can have a duration of, for example, about 10 hours to about 75 hours. The freeze-drying procedure may include a secondary drying step, for example at a temperature of about -10°C to about 40°C. The primary drying step can have a duration of, for example, about 1 hour to about 24 hours. During drying, the pressure in the freeze-drying chamber is adjusted to be lower than atmospheric pressure, e.g., about 10 mTorr. A vacuum is applied such that r is in the range of about 250 mTorr.

[0083] Other drying methods include spray drying, fluidized bed drying, dehumidification, and desiccation. n), and drying of filter cakes (N.P. Cheremisinoff ( 2000) Handbook of Chemical Preocessing Eq. uipment,butterworth Heinemann,Boston,MA (See ref.). Dehumidifiers are manufactured by Bry Air, Inc., Sunbury, Ohio, and and DST Seibu Giken, Wyomissing, PA. Rotary, conical, and shelf dryers are available (Mc Gill AirPressure LLC, Columbus, Ohio). In this mode, vacuum dryers remove water by exposing the material to reduced pressure, allowing evaporation to occur. The heat lost during the experiment is replaced by heat from a heat source or reservoir, such as a water bath. For example, these include materials found in commercially available rotary dryers. Rica gel desiccants, molecular sieve desiccants such as aluminosilicate and synthetic zeolite, and bentonite desiccants.

[0084] In some embodiments, reaction mixtures are prepared in the same manner as they are reconstituted for use. Dry in the same tank.

[0085] A lyophilized or otherwise solid or dry composition may contain, for example, less than 5% water by weight, less than 4% by weight %, less than 3% by weight, less than 2% by weight, less than 1.0% by weight, less than 0.5% by weight, less than 0.2% by weight Less than 0.1% by weight, less than 0.05% by weight, less than 0.02% by weight, less than 0.01% by weight In some embodiments, the solid composition has a low moisture content, such as less than about 5 mg. ~20g, 200mg~20g, 1g~20g, 5g~20g, 5mg ~1g, 5mg~500mg, 5mg~200mg, 5mg~100mg , about 5mg to about 50mg, about 5mg to about 20mg, about 5mg to about 10mg, about 10mg to About 200 mg, about 20 mg to about 200 mg, about 50 mg to about 200 mg, or about 100 It has a mass in the range of 100 mg to about 200 mg.

[0086] The lyophilized or otherwise solid or dry composition is stored prior to use. The storage period is The period may include the period during which the dry composition is stored at room temperature exposed to ambient air. , up to 3 hours, or up to 1.0 hour, up to 1.5 hours, up to 2.0 hours, up to 2.5 hours, max 3.5 hours, max 4.0 hours, max 5.0 hours, max 6.0 hours, max 8.0 The time may range anywhere from 100 to 200 hours, or from 90 to 180 minutes. The absolute humidity during storage must be at least 2.3 g of water per cubic meter of air, or 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 grams per cubic meter and up to an absolute humidity corresponding to about 95% relative humidity under storage conditions. Alternatively, the relative humidity may be, for example, about 40%, about 50%, about 60%, about 70%, about 80%, The relative humidity may be about 40% to about 95%, such as about 90% or about 95% relative humidity. stomach.

[0087] Storage also refers to a process in which the dry or solid composition is sealed and exposed to ambient air outside the seal. This storage period may include a longer period that qualitatively prevents the at least one week, at least one month, at least six months, at least one year, or at least two The period may be one year. A period of one month to two years is exemplary.

[0088] Storage temperature ranges for long-term storage or long-term or short-term stability studies include, for example: , 0~2℃, 0~4℃, 2~4℃, 2~6℃, 20℃, 25℃, 30℃, 40℃, 50℃ , 60℃, and under liquid nitrogen at -4 to -2℃, -6 to -2℃, -8 to -2℃, -10 to Examples of subzero temperatures include -2°C, -20°C, -40°C, -60°C, and -80°C. Typically, storage is in the range of about 4-8°C above the freezing point. Accelerated decomposition studies can be performed, e.g. , 1 hour, 2 hours, 4 hours, 24 hours, 2 days, 4 days, 8 days, 1 month, 3 months, 10 The incubation may be carried out at about 25° C., about 30° C., about 35° C., about 40° C., for a period such as 0 days. Conditions for storage or stability testing vary from above the freezing point to below the freezing point. The temperature may fluctuate as described above.

[0089] In some embodiments, the solution comprises water. In some embodiments, the solution comprises at least At least 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 55% by weight, 6 0% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight , 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, 99.5% by weight, 9 Contains 9.6%, 99.7%, 99.8%, or 99.9% water by weight. In some embodiments, the solution contains at least 10%, 20%, 30%, 40% by volume. vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol%, 75 vol%, 80% by volume, 85% by volume, 90% by volume, 95% by volume, 96% by volume, 97% by volume, 98% by volume %, 99% by volume, 99.5% by volume, 99.6% by volume, 99.7% by volume, 99.8% by volume, Or contains 99.9% water by volume.

[0090] In some embodiments, the solution comprises a polar organic solvent. The liquid may be at least 10%, 20%, 30%, 40%, 50%, 55%, Weight%, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, 99.5 %, 99.6%, 99.7%, 99.8%, or 99.9% by weight In some embodiments, the solution comprises at least 10% by volume, 20% by volume, or a mixture of both. %, 30% by volume, 40% by volume, 50% by volume, 55% by volume, 60% by volume, 65% by volume, 70 vol%, 75 vol%, 80 vol%, 85 vol%, 90 vol%, 95 vol%, 96 vol%, 97% by volume, 98% by volume, 99% by volume, 99.5% by volume, 99.6% by volume, 99.7% by volume %, 99.8%, or 99.9% by volume of a polar organic solvent in water. In some embodiments, the polar organic solvent comprises ethanol. In some embodiments, the polar organic solvent comprises , isopropanol. In some embodiments, the polar organic solvent comprises DMSO. In some embodiments, the polar organic solvent comprises glycerol.

[0091] In some embodiments, the solution comprises a non-polar organic solvent. The solution may be at least 10%, 20%, 30%, 40%, 50%, 5 5% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight , 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, 99. 5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight In some embodiments, the solution comprises at least 10% by volume, 20% by volume, of a non-polar organic solvent. vol%, 30 vol%, 40 vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70% by volume, 75% by volume, 80% by volume, 85% by volume, 90% by volume, 95% by volume, 96% by volume %, 97% by volume, 98% by volume, 99% by volume, 99.5% by volume, 99.6% by volume, 99.7 %, 99.8%, or 99.9% by volume of a non-polar organic solvent water.

[0092] In some embodiments, the total amount of solute in the solution is about 0.01% to about 50% (w / v). , about 0.05% to about 50% (w / v), about 0.1% to about 50% (w / v), about 0.2% to Approximately 50% (w / v), approximately 0.5% to approximately 50% (w / v), approximately 1% to approximately 50% (w / v), Approximately 2% to approximately 50% (w / v), approximately 5% to approximately 50% (w / v), approximately 10% to approximately 50% (w / v), about 0.01% to about 25% (w / v), about 0.01% to about 10% (w / v), about 0. 01%~approx. 5%(w / v), approx. 0.01%~approx. 2%(w / v), approx. 0.01%~approx. 1%( w / v), about 0.01% to about 0.1% (w / v), about 0.1% to about 1% (w / v), about 1 %~Approx. 2%(w / v), Approx. 2%~Approx. 5%(w / v), Approx. 5%~Approx. 10%(w / v), Approx. 1 0% to about 20% (w / v), or about 20% to about 50% (w / v). In an embodiment, the total amount of solute in the solution is about 0.01% to about 50% (w / w), about 0.05% ~50% (w / w), ~0.1%~50% (w / w), ~0.2%~50% (w / w), about 0.5% to about 50% (w / w), about 1% to about 50% (w / w), about 2% to about 50 %(w / w), about 5% to about 50%(w / w), about 10% to about 50%(w / w), about 0.0 1%~about 25%(w / w), about 0.01%~about 10%(w / w), about 0.01%~about 5% (w / w), about 0.01% to about 2% (w / w), about 0.01% to about 1% (w / w), about 0 0.01% to about 0.1% (w / w), about 0.1% to about 1% (w / w), about 1% to about 2% (w / w), about 2% to about 5% (w / w), about 5% to about 10% (w / w), about 10% to about 20% (w / w), or about 20% to about 50% (w / w). The total amount of solute in the solution is about 0.01% to about 50% (v / v), about 0.05% to about 50% (v / v), about 0.1% to about 50% (v / v), about 0.2% to about 50% (v / v), about 0.5 %~50%(v / v), approx.1%~50%(v / v), approx.2%~50%(v / v), Approximately 5% to approximately 50% (v / v), approximately 10% to approximately 50% (v / v), approximately 0.01% to approximately 25% (v / v), about 0.01% to about 10% (v / v), about 0.01% to about 5% (v / v), about 0.01%~Approx. 2%(v / v), Approx. 0.01%~Approx. 1%(v / v), Approx. 0.01%~Approx. 0 .1% (v / v), about 0.1% to about 1% (v / v), about 1% to about 2% (v / v), about 2% ~5% (v / v), ~10% (v / v), ~20% (v / v), or about 20% to about 50% (v / v).

[0093] Exemplary volumes of the solution include about 1 uL, about 5 uL, about 10 uL, about 20 uL, about 24 uL, and about 30 uL. uL, approx. 50uL, approx. 100uL, approx. 200uL, approx. 300uL, approx. 400uL, approx. 50 0uL, approx. 600uL, approx. 700uL, approx. 800uL, approx. 900uL, approx. 1,000uL (1 mL), about 2 mL, about 5 mL, about 10 mL, about 20 mL, and about 50 mL. The reconstituted composition may be formulated in the same volume, a lower volume, or a larger volume than the solution before drying. The lower volume may be about 90%, about 80%, about 60%, about 40% relative to the bulk reagent. %, about 20%, about 10%, or about 5%. The larger volume may be a bulk reagent. Approximately 120%, 140%, 160%, 180%, 200% (2x), approximately 4x, approximately 6x It may be about 8 times, about 10 times, or about 20 times.

[0094] In some embodiments, the solution comprises a bulking agent. In some embodiments, the solution comprises: In some embodiments, the solution is useful for performing a molecular assay. In some embodiments, the solution comprises at least one oligonucleotide. In some embodiments, the solution comprises a salt. In some embodiments, the solution comprises a In some embodiments, the solution comprises one or more nucleoside triphosphates. In some embodiments, the solution comprises dATP. , dGTP, dCTP, and at least one of dTTP and dUTP .

[0095] In some embodiments, the solution does not contain inorganic salts. contains about 5 mM to 0 mM of inorganic salt. Further exemplary concentration ranges of inorganic salt in the solution is about 4 mM to 0 mM, about 3 mM to 0 mM, about 2 mM to 0 mM, about 1 mM to 0 mM, or Additional common inactivation agents for amplification and detection reaction mixtures are approximately 500 μM to 0 mM. Some examples of inorganic salts include sodium, potassium, manganese, and magnesium. and chloride.

[0096] In some embodiments, the solution comprises 5 mM or less of an inorganic salt, and the inorganic salt comprises 1 micromolar or less of an inorganic salt. 0.373 μg / μL or less per liter, or 0.332 μg / μL or less, or 0 .292μg / μL or less by mass, and sodium chloride is present per microliter Quality of 0.292μg / μL or less, 0.146μg / μL or less, or 0.0μg / μL or sodium is present in an amount equal to or less than 0.115 μg / μL per microliter; Present at a mass of 0.057 μg / μL or less, or 0.0 μg / μL, or potassium chloride The concentration of 0.373 μg / μL or less per microliter and 0.186 μg / μL or less , or 0.0 μg / μL by mass, or potassium is present in 0.196μg / μL or less, 0.098μg / μL or less, or 0.0μg / μL Present by mass or chloride is 0.355 μg / μL or more per microliter Lower, 0.178μg / μL or less, 0.089μg / μL or less, or 0.0μg / μL It exists with a mass of

[0097] In some embodiments, the solution comprises 4 mM or less of an inorganic salt, and the inorganic salt comprises 1 micromolar or less of an inorganic salt. 0.298 μg / μL or less per liter, or 0.266 μg / μL or less, or 0 0.234 μg / μL by mass, and sodium chloride may be present in 0.234μg / μL or less, 0.117μg / μL or less, or 0.0μg / μL Sodium present by mass or less than 0.092 μg / μL per microliter , 0.046 μg / μL or less, or 0.0 μg / μL by mass, or potassium chloride The concentration of uranium is 0.298 μg / μL or less per microliter, and 0.149 μg / μL or less or 0.0 μg / μL, or potassium is present in a mass of 1 microliter 0.156μg / μL or less, 0.078μg / μL or less, or 0.0μg / μL or chloride is present in a mass of 0.284 μg / μL per microliter or less, 0.142μg / μL or less, 0.071μg / μL or less, or 0.0μg / μ It exists with a mass of L.

[0098] In some embodiments, the solution comprises 3 mM or less of an inorganic salt, and the inorganic salt comprises 1 micromolar or less of an inorganic salt. 0.224 μg / μL or less per liter, or 0.199 μg / μL or less, or 0 .175μg / μL or less by mass, sodium chloride is present per microliter Quality of 0.175μg / μL or less, 0.088μg / μL or less, or 0.0μg / μL or sodium is present in an amount equal to or less than 0.069 μg / μL per microliter; Present at a mass of 0.034 μg / μL or less, or 0.0 μg / μL, or potassium chloride The concentration of 0.224μg / μL or less per microliter and 0.112μg / μL or less , or 0.0 μg / μL by mass, or potassium is present in 0.117μg / μL or less, 0.059μg / μL or less, or 0.0μg / μL Present by mass or chloride is 0.213 μg / μL or more per microliter Lower, 0.107μg / μL or less, 0.053μg / μL or less, or 0.0μg / μL It exists with a mass of

[0099] In some embodiments, the solution comprises 2 mM or less of an inorganic salt, and the inorganic salt comprises 1 micromolar or less of an inorganic salt. 0.149 μg / μL or less per liter, or 0.133 μg / μL or less, or 0 .117μg / μL or less by mass, and sodium chloride is present per microliter Quality of 0.117μg / μL or less, 0.058μg / μL or less, or 0.0μg / μL or sodium is present in an amount equal to or less than 0.046 μg / μL per microliter; Present at a mass of 0.023 μg / μL or less, or 0.0 μg / μL, or potassium chloride The concentration of 0.149μg / μL or less per microliter and 0.075μg / μL or less , or 0.0 μg / μL by mass, or potassium is present in 0.078μg / μL or less, 0.039μg / μL or less, or 0.0μg / μL Present by mass or chloride is 0.142 μg / μL or more per microliter Lower, 0.071μg / μL or less, 0.036μg / μL or less, or 0.0μg / μL It exists with a mass of

[0100] In some embodiments, the solution comprises 1 mM or less of an inorganic salt, and the inorganic salt comprises 1 micromolar or less of an inorganic salt. 0.075 μg / μL or less per liter, or 0.066 μg / μL or less, or 0 Sodium chloride is present in a mass of 0.058 μg / μL or less per microliter. Quality of 0.058μg / μL or less, 0.029μg / μL or less, or 0.0μg / μL or sodium is present in an amount of 0.023 μg / μL or less per microliter; Present at a mass of 0.011 μg / μL or less, or 0.0 μg / μL, or potassium chloride The concentration of 0.075μg / μL or less per microliter and 0.037μg / μL or less , or 0.0 μg / μL by mass, or potassium is present in 0.039μg / μL or less, 0.020μg / μL or less, or 0.0μg / μL Present by mass or chloride is 0.071 μg / μL or more per microliter Lower, 0.036μg / μL or less, 0.018μg / μL or less, or 0.0μg / μL It exists with a mass of

[0101] In some embodiments, the solution comprises 500 μM or less of an inorganic salt, and the inorganic salt is 1 μM or less. Not more than 0.037 μg / μL per chlorite, or not more than 0.033 μg / μL, or is present in a mass of 0.029 μg / μL or less, and sodium chloride is present in a mass of 0.029 μg / μL or less. 0.029μg / μL or less, 0.015μg / μL or less, or 0.0μg / μL or sodium is present in a mass of 0.011 μg / μL or less per microliter. or less than 0.006 μg / μL, or 0.0 μg / μL by mass, or potassium chloride Sodium is less than 0.037μg / μL and less than 0.019μg / μL per microliter. Potassium is present in a mass of 0.0 μg / μL or less, or in a mass of 1 microliter per unit of sample: 0.020μg / μL or less, 0.010μg / μL or less, or 0.0μg / μ L or chloride is present at a mass of 0.036 μg / μL L or less, 0.018μg / μL or less, 0.009μg / μL or less, or 0.0μg / It is present in a mass of μL.

[0102] In some embodiments, the solution comprises about 5 mM to about 500 μM of an inorganic salt, , with a mass of about 0.373 μg / μL to about 0.029 μg / μL per microliter The sodium chloride concentration is 0.292 μg / μL to about 0.0 29 μg / μL by mass, and sodium is present at 0.115 μg / μL. 0.006 μg / μL, and potassium chloride is present in a volume of 1 microliter. The amount of potassium is about 0.373 μg / μL to about 0.019 μg / μL per unit weight. It exists in a mass range of 0.196 μg / μL to about 0.010 μg / μL per microliter. The chloride concentration ranges from 0.355 μg / μL to approximately 0.009 μg / μL. The inorganic salt is present in a mass of about 0.373 μg / μL per microliter. ~ 0.029 μg / μL by mass, and sodium chloride is present in The inorganic salt may be present at a mass of about 0 μg / μL per microliter, or the inorganic salt may be present at a mass of about 0.373μg / μL to about 0.029μg / μL, and potassium chloride is present at 1 It is present at a mass of approximately 0 μg / μL per microliter.

[0103] In some embodiments, at least one of the one or more nucleoside triphosphates is In the solution, in an amount ranging from about 0.1 mM to about 0.3 mM or from about 0.2 mM to about 0.6 mM exist.

[0104] In some embodiments, the solution contains a pharma- ceutically active agent, e.g., one or more single units. In some embodiments, the solution contains a pharma- ceutically acceptable dose of a pharma- ceutically active agent. The present invention further includes salts thereof.

[0105] In some embodiments, the solution is sufficient to perform a nucleic acid synthesis or amplification assay. "Reagents sufficient to perform a nucleic acid synthesis or amplification assay" includes polymerase chain reaction (PCR) and / or amplification assays. To carry out nucleic acid synthesis, a nucleic acid synthesis kit is provided, such as a nucleic acid synthesis kit, a suitable divalent cation salt, and nucleotide triphosphates. It means a set of reagents other than a solvent and a sample or a template nucleic acid that can be used for the purpose of Reagent sets such as these allow for PCR, RT-PCR, and It can be customized for various types of amplification, including transcription-mediated amplification.

[0106] In some embodiments, the solution comprises a capture probe or a hydrolysis probe. In some embodiments, the solution includes an RNase inhibitor. The ase inhibitor is present in the solution at a concentration of about 0.12 U / μL to about 0.20 U / μL. In some embodiments, the solution includes a chelating agent. The inhibitor is present in the solution at a concentration of 1.5 mM to 2.0 mM. The solution includes a detergent.

[0107] In some embodiments, a solution according to the present disclosure (sometimes referred to as a pre-lyophilization mixture) is (a) polymerase; nucleotides for use in amplification and detection reactions; organic buffers; , for example, Tris; and trehalose or raffinose or combinations thereof. The solution may also contain one, two, three or all of the following fillers: The solution may or may not contain nucleic acids. It contains a reverse transcriptase, a chelating agent, and an RNase inhibitor. The composition may further include a harmful agent.

[0108] Nucleotides for incorporation into an amplification reaction are typically provided as dNTPs. Exemplary concentrations for NTPs are 0.1-0.3 mM dATP, 0.1-0.3 mM dGTP, 0.1-0.3 mM dCTP, 0.2-0.6 mM dTTP, 0.2-0 0.6 mM dUTP, preferably about 0.2 mM dATP, about 0.2 mM dGTP, about 0.2 mM dCTP, and 0.4 mM dTTP or dUTP.

[0109] An exemplary solution contains 0.1 to 0.3 mM, more preferably 0.2 mM dATP, dGT P, and dCTP, and 0.2 to 0.6 mM, more preferably 0.4 mM dUT P or dTTP, and 0.3–0.8 U / µL of polymerase. In the composition, the polymerase is a hot-start Taq polymerase. In the composition, the polymerase is GoTaq® MDx Hot Star Some compositions also contain 0.12-0.20 U / μL of RNA polymerase. Some compositions also optionally contain 1.5 to 20% riboflavin (TM) RNAse inhibitor. Such compositions also contain 0.16 to 0.32 M trehalose. and 1.5 to 2.0 mM EDTA, and the polymerase is 0.3 to 0.45 U / μL of Taq.

[0110] The present disclosure provides reagents for PCR reactions, including real-time PCR reactions. al-time PCR Handbook, Life Technologies(2 014), Kutyavin et al (2000) Nucleic Acids R es.28:655, Afonina et al (2002) Biotechniqu es.32:940). In real-time PCR, magnesium salts are typically used at about Used at a final concentration of 3–6 mM (Real-time PCR Handbook, In some embodiments, the present disclosure provides a method for multiplex PCR reactions, i.e. To provide a reagent where multiple primer pairs are provided for amplification and detection of multiple targets. The present disclosure provides primers and probes for PCR reactions. and / or the probe comprises a target hybridizing sequence and a non-target hybridizing sequence, one or more of a nucleotide analog, a detectable moiety, and a non-nucleotide linker (see, for example, WO2010 / 151566 and WO2013 / 1267 93). In some embodiments, one or more primers or probes is suitable for hybridization or under stringent conditions with the target nucleic acid , hybridize to template nucleic acid, or sample nucleic acid. Thermocyclers are available. (Applied Biosystems ProFlex® PCR System tem and Veriti™ Thermal Cycler. Quantitate PCR products. A gel scanner for analyzing the gels is available (Agilent® 2100 B ioanalyzer®, Bio-Rad® densitometer).

[0111] After the solution is formed, it may be left at room temperature for a significant period of time before drying. up to 8 hours before the start of the tapping step or up to 1 hour before the start of the drying step. Up to 2 hours, up to 4 hours, up to 6 hours, up to 10 hours, up to 12 hours, or up to 14:00 Including significant amounts of salt in the solution along with other reagents, such as PCR reagents, can During this incubation period, unwanted hybridization products and other By-products may occur. Such undesired hybridization products and the by-product is a solution essentially free of inorganic salts, e.g., having less than about 5 mM inorganic salts. This can be reduced or eliminated by forming

[0112] The presence of inorganic salts in a solution can result in some of the following undesirable properties, depending on the other components of the solution: One or more of these may occur. Nucleic acids can hybridize together and hybridize The ions include potassium, sodium, manganese, magnesium, and / or chloride. It is stimulated by the presence of inorganic ions from any salt, such as manganese and magnesium. Even in the presence of inorganic divalent cations, undesirable enzyme activity such as polymerase processivity was not observed. Such undesirable activity can occur due to non-hot start enzymes and hot start enzymes. Nucleic acid hybridization and enzyme activity in the presence of salts As a result, undesirable by-products may begin to form. In addition, inorganic salts Being hygroscopic, the solid composition can draw atmospheric moisture into it. Rehydration of the solid composition can be achieved after storage. This can reduce enzyme stability and allow for the formation of additional spurious by-products.

[0113] In some embodiments, a solid composition is prepared by drying a solution according to the present disclosure. Thus, a solid composition according to the present disclosure is formed by the addition of a solution according to the present disclosure. For example, in some embodiments, the solid composition may contain any solute that can be dissolved in water. In some embodiments, the solid composition comprises one or more enzymes. In an embodiment, the solid composition comprises at least one oligonucleotide useful for performing a molecular assay. In some embodiments, the solid composition comprises a buffer. In some embodiments, the solid composition comprises a salt. In some embodiments, the solid composition comprises one or more nucleoside triphosphates. In some embodiments, the solid composition comprises one or more deoxynucleosides. In some embodiments, the solid composition comprises dATP, dGTP, dCT P, and at least one of dTTP and dUTP. In some embodiments, the solid composition comprises a capture probe or a hydrolysis probe. In some embodiments, the solid composition comprises an RNase inhibitor. comprises a chelating agent. In some embodiments, the solid composition comprises a detergent.

[0114] In some embodiments, the solid composition is useful for performing a nucleic acid synthesis or amplification assay. In some embodiments, the solid composition comprises a polymerase; an amplification and Nucleotides for use in detection reactions; organic buffers, e.g., Tris; and trehalose. One or two of the following fillers: sucrose or raffinose or a combination thereof , 3, or all of the above. The solution may or may not also contain one or more nucleic acids. The solution may further comprise a reverse transcriptase, a chelating agent, and an RNase inhibitor.

[0115] The solid composition provides a single unit dose (SUD), or optionally two or more SUDs. A single unit dose may contain reagents for performing amplification and / or detection reactions on a single or less than one sample. A single unit dose is a collection of reagents necessary to carry out a reaction. As referred to herein, a single unit dose can refer to a single sample. It is not necessary to include all of the reagents necessary to carry out the amplification and / or detection reactions. It is noteworthy that a single unit dose is sufficient to carry out the amplification and / or detection reaction. Similarly, a single unit dose may lack the reagents necessary for amplification and / or detection. It may contain insufficient amounts of reagents to carry out a reaction. By way of example only, a dry single unit The dose pellet may contain the appropriate units of Taq polymerase to carry out an amplification reaction. However, magnesium may be absent. In such an example, magnesium may be present, for example, in the form of re A constituent liquid may be added to the dry single unit dose pellets. Thus, a dry single unit dose may contain insufficient amounts of dNTPs to perform an amplification reaction. In such an instance, the remainder of the dNTPs may be reconstituted, for example, by a reconstitution liquid, into a dried single aliquot. A person skilled in the art, having access to this disclosure, would recognize that these examples are non-limiting. This allows for the easy production of SUDs and dry pellet SUDs with a variety of compositions. As used herein, the phrase "containing a single unit dose" and its equivalents are intended to mean Other grammatical forms do not embrace unlimited quantities, i.e., solutions, compositions, etc., containing a single unit dose. No composition, or concave surface (eg, well) contains more than one SUD.

[0116] In some embodiments, the solid composition contains a pharma- ceutical active agent, e.g., one or more monosaccharides. In some embodiments, the solid composition comprises a unit dose of a pharma- ceutical active agent. The present invention further includes physiologically acceptable salts.

[0117] In some embodiments, the solid composition is prepared by drying a solution containing 5 mM or less of an inorganic salt. The weight percentage of the inorganic salt relative to the weight of the solid composition is 0.311%. or less, 0.277% or less, or 0.244% or less. For example, the solution may be 4 mM or less of inorganic salt, and the weight percent of the inorganic salt relative to the weight of the solid composition is 0. 249% or less, 0.222% or less, or 0.195% or less. The solution is 3 mM or less of inorganic salt, and the weight percent of the inorganic salt relative to the weight of the solid composition is 0. 186% or less, 0.166% or less, or 0.146% or less. The solution is 2 mM or less of inorganic salt, and the weight percent of the inorganic salt relative to the weight of the solid composition is 0. 124% or less, 0.111% or less, or 0.097% or less. The solution is 1 mM or less. of inorganic salt, and the weight percent of the inorganic salt relative to the weight of the solid composition is 0. The solution is 500 μM The following inorganic salts can be included, the weight percentage of the inorganic salt relative to the weight of the solid composition being: The solution is 5 mM The solid composition may contain up to 500 μM of an inorganic salt, and the percentage by mass of the inorganic salt relative to the mass of the solid composition may be In some embodiments, the solid composition contains about 0.311% to about 0.024%. The weight percent of sodium chloride relative to the weight of the solid composition is about 0% or The mass percentage of potassium chloride relative to the mass is about 0%. Also provided is a vessel containing one dry single unit dose of the compound. Multiwell plates are also provided, each having at least 2, 3, 4, 5, 6, 10, 12 wells. Each, or all, contains a dry single unit dose of one of the solid compositions described above.

[0118] In some embodiments, a reconstitution liquid is used or provided. For example, the reconstitution liquid may be For example, the solid composition on the plasma-treated surface may be added. In some embodiments, A reconstitution liquid is provided in the kit.

[0119] In some embodiments, the reconstitution liquid comprises water. The liquid may be at least 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 5 5% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight , 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, 99. 5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight In some embodiments, the reconstitution liquid comprises at least 10% by volume, 20% by volume, , 30 vol%, 40 vol%, 50 vol%, 55 vol%, 60 vol%, 65 vol%, 70 vol vol%, 75 vol%, 80 vol%, 85 vol%, 90 vol%, 95 vol%, 96 vol%, 9 7% by volume, 98% by volume, 99% by volume, 99.5% by volume, 99.6% by volume, 99.7% by volume , 99.8%, or 99.9% water by volume.

[0120] In some embodiments, the reconstitution liquid comprises a polar organic solvent. The reconstituted liquid may contain at least 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, Weight%, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight %, 99.5% by weight, 99.6% by weight, 99.7% by weight, 99.8% by weight, or 99. 9% by weight of a polar organic solvent. In some embodiments, the reconstitution liquid comprises at least 1 0% by volume, 20% by volume, 30% by volume, 40% by volume, 50% by volume, 55% by volume, 60% by volume , 65 vol%, 70 vol%, 75 vol%, 80 vol%, 85 vol%, 90 vol%, 95 vol %, 96% by volume, 97% by volume, 98% by volume, 99% by volume, 99.5% by volume, 99.6% by volume %, 99.7%, 99.8%, or 99.9% by volume of polar organic solvent water In some embodiments, the polar organic solvent comprises ethanol. In some embodiments, the polar organic solvent comprises isopropanol. comprises DMSO. In some embodiments, the polar organic solvent comprises glycerol.

[0121] In some embodiments, the reconstitution liquid comprises a non-polar organic solvent. In the reconstituted liquid, the reconstituted liquid is at least 10% by weight, 20% by weight, 30% by weight, 40% by weight, 5 0% by weight, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight , 85% by weight, 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight %, 99.5%, 99.6%, 99.7%, 99.8%, or 99 In some embodiments, the reconstitution liquid comprises at least 0.9% by weight of a non-polar organic solvent. Also 10 volume%, 20 volume%, 30 volume%, 40 volume%, 50 volume%, 55 volume%, 60 volume %, 65 vol%, 70 vol%, 75 vol%, 80 vol%, 85 vol%, 90 vol%, 9 5% by volume, 96% by volume, 97% by volume, 98% by volume, 99% by volume, 99.5% by volume, 99. 6%, 99.7%, 99.8%, or 99.9% by volume of non-polar polar organic The solvent includes water.

[0122] In some embodiments, the reconstitution liquid comprises a bulking agent. The reconstituted liquid comprises one or more enzymes. In some embodiments, the reconstituted liquid comprises a molecular assay. In some embodiments, the nucleic acid sequence includes at least one oligonucleotide useful for carrying out the nucleic acid sequence. In some embodiments, the reconstitution liquid comprises a buffer. In some embodiments, the reconstitution liquid comprises a salt. In some embodiments, the reconstitution liquid is free of inorganic salts. In some embodiments, the reconstitution liquid comprises one or more nucleoside triphosphates. The reconstitution liquid comprises one or more deoxynucleoside triphosphates. The reconstituted liquid contains dATP, dGTP, dCTP, as well as dTTP and dUTP. Including at least one of them.

[0123] In some embodiments, the reconstituted liquid is useful for performing a nucleic acid synthesis or amplification assay. Contains sufficient reagents.

[0124] In some embodiments, the reconstitution liquid comprises a capture probe or a hydrolysis probe. In some embodiments, the reconstitution liquid comprises an RNase inhibitor. In some embodiments, the reconstitution liquid comprises a chelating agent. Contains agents.

[0125] In some embodiments, the reconstitution liquid is free or substantially free of organic solutes. In some embodiments, the reconstituted liquid has a molecular weight greater than about 1 kDa. In some embodiments, the reconstitution step is carried out in a manner that is free or substantially free of organic solutes. The liquid contains no or substantially no organic solutes having a molecular weight greater than about 10 kDa. "Substantially free" means that the referenced substance, if present, is It may affect the suitability of the resulting composition for the appropriate purpose, e.g., reconstitution, detection, amplification, etc. This means that the level is low enough so that

[0126] In some embodiments, the reconstitution fluid comprises 3.8-4.4 mM MgCl2 in water, and and 50-80 mM KCl. The reconstitution fluid contains, among other components, 0. 0.012-0.020% methylparaben, 0.006-0.010% propylparaben , and / or 0.26% absolute ethanol.

[0127] In some embodiments, the reconstitution liquid comprises a pharma- ceutically acceptable salt. In embodiments, reconstituted liquid and solid compositions (e.g., solid compositions containing a pharma- ceutically active agent) are The combination of the two substances (compounds) may be used to produce, for example, a pharma- ceutically acceptable mixture containing a single unit dose of a pharma- ceutically active agent. To provide a composition which is acceptable.

[0128] The reconstitution time is the time period after the solid composition is contacted with an aqueous solution suitable for the intended use of the solid composition. Less than 1 second, less than 2 seconds, less than 5 seconds, less than 10 seconds, less than 15 seconds, less than 20 seconds, less than 50 seconds, The contacting may be for less than 60 seconds (1 minute) or less, and may optionally be performed by shaking, tapping, vortexing, etc. shaking, rocking, inserting and removing pipette tips, or folding or squeezing malleable vials The reconstitution time is facilitated by either: The reconstituted liquid is used to prepare the 100% 15 ... The composition may be measured either in a warm solution at 17°C or in a dry solution at 17°C. Typically, the dry composition is removed from the refrigerator and then cooled. The environment (room temperature) for any of these procedures should be kept cool. ) is typically at ambient temperature or about 21°C to 25°C, e.g. 23°C. The time point at which it is determined that the substance has been completely solubilized may be, for example, the time point at which it is determined that the substance has been completely solubilized. Complete solubilization may be indicated, for example, by the absence of turbidity or a Schlieren pattern of It may be determined by visual inspection, where the absence of any visible solubilization is a measure of complete solubilization. Complete solubilization can be determined by optical instruments, such as machines that measure light scattering.

[0129] Optional additional components (e.g., in the solution, solid composition, or reconstituted liquid) include: PCR reagents, detergents, primers, probes, templates, methylparaben, and propionyl These include, but are not limited to, any of the following: methylparaben; Typical concentrations are 0.01 to 0.024% by weight, for example about 0.016%, or about 0. 010%, approximately 0.014%, approximately 0.016%, approximately 0.020%, approximately 0.024%, or The range bounded by these values ​​is arbitrary. Typical concentrations of propylparaben The range is 0.002-0.016% or 0.008%, or about 0.002%, about 0.004%, approx. 0.006%, approx. 0.008%, approx. 0.010%, approx. 0.012%, approx. 0.014%, approximately 0.016%, or any range bounded by these values It is.

[0130] In some embodiments, the bulking agent (e.g., present in a solution or solid composition) is Exemplary bulking agents include trehalose or raffinose or combinations thereof. Other fillers that may be used include sucrose, mannitol, trehalose, Sucrose + mannitol, sucrose + mannitol, sucrose + glycine, and hydrochloride Cleland et al. (2001) J. Pha rm.Sci.90:310, Meyer et al (2009) Eur.J.Pha rm.Sci.38:29, Webb et al (2003) J.Pharm.Sci .92:715, Garzon Rodrigues et al (2004) J.Ph. arm.Sci.93:684, Qiu et al (2012) Int.J.Phar maceuticals.437:51), Van Dijk-Wolthuis et al. See, e.g., et al (1997) Polymer. 38:6235 6242. Hydroxyethyl starch is classified as hetastarch, hexastarch, pentastarch, and tetrastarch. Classified as a starch (see, e.g., WO 2014 / 099198 to Chow). The filler is preferably 0.16M to 0.32M, or 0.04 to 0.12M. M, 0.08~0.16M, 0.12~0.20M, 0.16~0.24M, 0.20~ 0.28M, 0.24~0.32M, 0.28~0.36M, 0.32~0.40M, or any combination of the aforementioned ranges, such as 0.08 to 0.24 M. In some embodiments, the bulking agent is sucrose, mannitol, glycine, hydroxyethyl ester. At least one of the following: starch, raffinose, or trehalose. In some embodiments, the bulking agent comprises trehalose.

[0131] In some embodiments, one or more enzymes (e.g., in a solution, solid composition, or reconstituted (present in liquids) are DNA polymerase, RNA polymerase, ligase, and kinase , phosphatases, proteases, exonucleases, and endonucleases In some embodiments, the one or more enzymes include at least one of the following: In some embodiments, the thermostable DNA polymerase comprises a Hot A polymerase. It is a start-up thermostable DNA polymerase or is conjugated to an antibody. In some embodiments, the one or more enzymes are present at about 0.20 U / uL to about 0.72 U / uL or The DNA-dependent polymerase present in the solution at a concentration of about 0.1 U / uL to about 0.6 U / uL The enzyme may include a ribozyme, ... reverse transcriptase, or other enzymes.

[0132] DNA polymerase enzymes can be commercially available or prepared by the user. An example of a polymerase enzyme is Taq polymerase (Ge (Rumtown, MD, Catalog No. 201203). An example of such a polymerase is GoTaq® G2 Flexi DNA polymerase (Promega). (M7801), available commercially from the University of Pennsylvania, Madison, WI. Other DNA polymerases that have been reported include Tth DNA polymerase (e.g., Sig Ma-Aldrich, St. Louis, MO, Catalog Number 11480022001 ), and Phusion® High-Fidelity DNA Polymerase Chimeric DNA polymerase such as ribosomes (NEB, Ipswich, MA, Catalog No. M0530S) These include, but are not limited to, hot-start DNA polymerases. Enzymes are also commercially available. For example, Taq polymerase is available as GoTaq® Hot Commercially available as Start Polymerase (Promega, catalog number M5001) GoTaq® Hot Start Polymerase is an antibody-mediated hot start enzyme. The Taq polymerase is bound to an antibody that blocks the polymerase activity. The cleavage antibody is denatured using high heat, thus denaturing the antibody during the initial heating step of the PCR reaction. The enzyme is denatured and polymerase activity is restored. Along with the hot start method, various antibodies, e.g. For example, TAQSTART antibody (Clontech Laboratories, Mount (Ain View, CA, Catalog No. R028A) can be used. Other hot-start polymerase enzymes, including mediated hot-start polymerases, are available. Equivalent polymerases and antibodies are available from a variety of commercial sources. or may be prepared by the user.

[0133] Reverse transcriptases can be commercially available or prepared by the user. Examples of enzymes include MMLV (Maloney Murine Leukemia Virus) reverse transcriptase and Sup erScript® III reverse transcriptase (e.g., ThermoFisher Scientific, Carlsbad, CA, Catalog No. 28025-013 and and 18080-044), MMLV RT (Sigma-Aldrich, Cat. No. M1302), AMV reverse transcriptase (NEB, Ipswich, MA, Cat. No. M02 77S), and GoScript™ reverse transcriptase (Promega, catalog no. No. A50003), but are not limited to GoScript reverse transcriptase. for the synthesis of first strand cDNA optimized for reverse transcriptase and quantitative PCR amplification Equivalent reverse transcriptases and reagents are available from a variety of commercial sources. This may be possible or may be adjusted by the user.

[0134] Exemplary concentrations of DNA polymerase enzyme in a single unit dose are 0.01 to 1.0 U / μL. For example, 0.32 U / μL, or 0.4 U / μL, or 0.72 U / μL, or 0.32-0.4 U / μL, or 0.4-0.72 U / μL, or 0.05- 0.3U / μL, or 0.8-1.0U / μL. One unit of DNA polymerase is 74 required to catalyze the incorporation of 10 nmoles of dNTPs into acid-insoluble material at 30 min at °C Exemplary concentrations of reverse transcriptase in a single unit dose are 0. 0.01U / μL to 1.0U / μL. One unit of reverse transcriptase is the enzyme that converts the The enzyme required to catalyze the transfer of 1 nmol of deoxynucleotide to a precipitable substance. It is defined as an elementary quantity.

[0135] In some embodiments, the oligonucleotide(s) (e.g., solution, solid composition) , or present in the reconstitution liquid) may be one or more amplification oligomers (e.g., primers , promoter-primer), capture probe, detection probe, Taqman probe, positive In some embodiments, the oligonucleotides are a positive control template, a positive control template, and a negative control template. In some embodiments, the label is a fluorescent, chemical In some embodiments, the label is a nucleoside. It is not an alkyl group, a phosphate, a carbohydrate, or a nucleobase.

[0136] In some embodiments, sufficient reagents (e.g., For example, in a solution, solid composition, or reconstituted liquid, the primer, promoter, A primer or primer pair (optionally including a promoter-primer) is then added. In some embodiments, sufficient reagents are included to perform a nucleic acid synthesis or amplification assay. The medicament may further comprise one or more of a buffering agent, stabilizer, preservative, or other auxiliary substance.

[0137] In some embodiments, a buffer (e.g., in a solution, solid composition, or reconstitution liquid) is used. In some embodiments, the buffer is an inorganic buffer. do.

[0138] An exemplary organic buffer is Tris. Solutions, solid compositions, or reconstituted liquids of the present disclosure Alternative organic buffers that can be incorporated include phosphate, citrate, acetate, CHES , histidine, and HEPES, MES, MOPS, tricine, and glycine amines. Other organic buffers include Good's buffers such as dimethylformamide, ... Examples include succinate, citrate, gluconate, and phosphate. In embodiments, the buffer has a pH range of about 5.5 to about 7.0 or about 6.0 to about 7.5, e.g. For example, a pH of about 6.5 is effective. Examples of buffers that control the pH within this range include cobalt. Succinates (such as sodium succinate), gluconates, histidine, citrates, and Other organic acid buffers are included.

[0139] In some embodiments, the chelating agent (e.g., a solution, a solid composition, or a reconstituted liquid EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetate), EDDS (Ethylenediamine-N,N'-disuccinic acid), MGDA (Methyl glycine diacetic acid), or DTPA (diethylenetriaminepentaacetic acid). An exemplary concentration of the chelating agent is about 1 mM to 2.5 mM.

[0140] In some embodiments, a detergent (e.g., present in a solution, solid composition, or reconstituted liquid) The detergents present are nonionic, cationic, anionic, or zwitterionic. Agents are available from many commercial vendors (e.g., Geno Technology, Inc. ionic (cationic or anionic), available from Sigma-Aldrich Co., St. Louis, MO; Nonionic and zwitterionic detergents. Examples include lithium lauryl sulfate. , amprolium hydrochloride, benzalkonium chloride, choline p-toluenesulfonate salt, Dodecyltrimethylammonium chloride, 3-[(3-cholamidopropyl)dimethyla ammonium io]-1-propanesulfonate, ethylhexadecyldimethylammonium bromide amide, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride Dodecyl sulfate, sodium dodecyl sulfate, hexadecyltrimethylammonium p-toluenesulfonate nate, luviquat(trademark), methylbenzethonium chloride, myristyl trime ethylammonium bromide, N,N',N'-polyoxyethylene (10)-N-tallow 1,3-Diaminopropane Liquid, Oxyphenonium Bromide, Tetraheptyl Ammonium ammonium bromide, tetraheptylammonium bromide, tetrakis(decyl)ammonium , Tricaprylylmethylammonium chloride, Amidosulfobetaine-1 6. Tridodecylmethylammonium chloride, trimethyloctadecylammonium bromide Nonidet P-40 (registered trademark), Tween-20 (registered trademark), Twe EN-80 (registered trademark), Brij-35 (registered trademark), Triton X-100 (registered trademark) Trademarks include, but are not limited to:

[0141] RNase inhibitor proteins bind non-covalently to RNases in a 1:1 ratio. Natural and recombinant 5-aminobutyric acid inhibitors that inhibit the RNase A family and human placental RNase 0 kDa protein (Promega Corp., Madison, WI). otella-Estrada et al (2001) Cancer Gene Th er.8:278, Polakowski et al (1992) EXS.61:42 8. Exemplary concentrations of RNase inhibitors are about 0.04 U / μL to about 0.4 One unit is the amount of ribonuclease A required to inhibit the activity of 5 ng of ribonuclease A by 50%. Activity is defined as the amount of RNasin® ribonuclease inhibitor required. Inhibition of the hydrolysis of cytidine 2',3'-cyclic monophosphate by ribonuclease A It is measured by.

[0142] In some embodiments, the solution is dispensed onto a surface prior to the drying step. In some embodiments, prior to the dispensing step, the surface is plasma treated to form a plasma treated surface. Form.

[0143] In some embodiments, the solid composition is within a concave surface of the plasma-treated surface. In some embodiments, the solid composition is in a vessel that includes a plasma-treated surface. In some embodiments, the vessel comprises a tube. In some embodiments, the vessel comprises a multi-well vessel. In some embodiments, the volume of the vessel, tube, or well is about 40 In some embodiments, after the drying step, the solid composition is The composition is sealed within a chamber containing the plasma-treated surface.

[0144] As mentioned above, certain embodiments include multi-well plates containing two or more wells. In some embodiments, the two or more wells have low water vapor transmission rate, low thermal conductivity, Includes walls constructed from materials that have optical transparency, low autofluorescence, or a combination thereof In one aspect, one or more of the wells comprises a cone-shaped wall. In some embodiments, two One or more wells are used to carry out a PCR amplification reaction on the reaction mixture contained within the wells. In some embodiments, the wall is configured to fit a PCR thermal cycler for In the example, two or more wells are used to perform PCR, TMA, or other nucleic acid amplification reactions. The thermally conductive tube may include a wall configured to fit into the thermally conductive tube receiving area of ​​the device. In some embodiments, two or more of the wells include openings for accessing the chambers of the wells. In some embodiments, two or more wells each have a sealing member to seal the opening of the associated well. In some embodiments, the opening of each of the two or more wells includes a cap. In some embodiments, the container is sealed with a cap that is a water vapor permeable foil. Each opening of the well is sealed with a cap that is an elastomeric material with low water vapor transmission rate. In one embodiment, the multi-well plate comprises two or more wells as described herein. Each chamber of the two or more wells is a dry, single cell suspension containing a polymerase enzyme and an inorganic salt. The unit dose composition comprises a weight percentage of inorganic salt relative to the weight of the pellet of about 0.3 In one embodiment, the multi-well plate comprises a multi-well plate having a concentration of 0.11% to 0.024%. and two or more wells in which a reverse transcriptase and an inorganic phosphate buffer are present, and a salt, and the weight ratio of the inorganic salt to the weight of the pellet is In one embodiment, the concentration of the cellulose acetate solution in the multi-well plate is about 0.311% to 0.024%. comprises two or more wells as described herein, and provides a dry, single-unit dose composition comprising a polymerase enzyme, a reverse transcriptase enzyme, and an inorganic salt. The mass percentage of inorganic salts relative to the mass of the pellet is about 0.311% to 0.024%. %.

[0145] The solid compositions provided herein may be reconstituted with a reconstitution liquid as described above and then one or more The sample to be analyzed may be used prior to reconstitution, simultaneously with reconstitution, In some embodiments, the reconstitution step may be performed after the addition of the glycerol to the solid composition. The entire composition after reconstitution is used to combine with the sample, where the reconstituted liquid / sample The relative volumes are, for example, about 9.9 / 0.1, 9.8 / 0.2, 9.5 / 0.5, 9 / 1, 8 It could be / 2, 7 / 3, 6 / 4, 5 / 5, etc.

[0146] For example, in some embodiments, a nucleic acid is added, the nucleic acid comprising at least one nucleic acid modification. In some embodiments, the nucleic acid modification or hybridization reaction is carried out. In some embodiments, the hybridization reaction comprises nucleic acid synthesis or amplification. The acid modification or hybridization reaction hybridizes the probe to the nucleic acid to form the hybrid In some embodiments, the method includes forming a hybridized complex. detecting hybridized complexes or nuclear lysis of the lobes.

[0147] Unless otherwise stated, the concentration of a reagent in a solution or reconstituted composition is, for example, 0.0% ( Reagents omitted), 0.001%, 0.004%, 0.008%, 0.0012%, 0. 0016%, 0.0020%, 0.0030%, 0.0040%, 0.0050%, 0. 0060%, 0.0080%, 0.01%, 0.02%, 0.04%, 0.06%, 0. 1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2%, 3 %, 4%, 5%, etc. Also provided are reagents in a range involving any two of the above concentrations greater than or equal to 100 mg / kg.

[0148] Carrying out the steps described above, such as adding a reconstitution liquid to the solid composition on the plasma-treated surface. In some embodiments, an automated sample collection and handling device may be used to collect and analyze the samples. Additional substances, such as samples or templates, enzymes, or salts, may be added simultaneously with or in conjunction with the reconstitution. The reaction mixture is then added sequentially using an automated sample handling device. The reaction can be carried out in an automated sample handling device. In embodiments, the automated sample collection and handling device is capable of performing multiple assays simultaneously, e.g., PCR. A device capable of carrying out the reaction, transcription-mediated amplification, and target capture hybridization. devices such as the Hologic® Panther instrument (Hologic, nc., MA) equipped with pipettors, mixers, incubators, and washing stations. It is a robotic device that

[0149] When one or more enzymes are present, the essential absence of inorganic salts is essential to the bulk composition prior to drying. Enzyme activity during storage of the reagent, during short-term storage of the dry composition before sealing, and during long-term storage after sealing In some embodiments, the enzyme activity after total storage may be reduced. The stability is less than that of the control sample stored immediately before the start of storage or under optimal conditions. At least 99%, at least 98%, at least 95%, at least 90%, at least 80% %, at least 75%, at least 70%, at least 60%, at least 50%, at least at least 40%, at least 30%, at least 20%, and the boundaries between these percentages The boundary is a defined area.

[0150] The stability of a composition can be determined, for example, by the activity (i.e., rate or yield of amplification) or the by-products. The lack of stability can be assessed after reconstitution of the dried product from the formation of This may be due to loss of activity or formation of by-products during storage. The formation or formation may be an absolute or relative measure. If it is relative, a basis for comparison is provided. The solution may be a bulk reagent mix prior to drying and reconstitution, or may not be specifically intended for testing. A different control reconstitution mixture (e.g., Mg 2+ or another salt thereof). is evaluated by real-time amplification rate, or final yield of amplified product, or hit rate. The by-products are gel electrophoresis, gel scanners, agarose gels, capillary electrophoresis The assay may be performed by one or more of the following:

[0151] The activity of the reconstituted amplification mixture (where necessary for any differences due to reconstitution of different volumes) (corrected if necessary) is preferably within 75, 80, 85, 90, or 95%. or indistinguishable within experimental error from the activity of the bulk reagent before drying. By-products present in the mixture (corrected if necessary for any differences due to reconstitution of different volumes) (corrected) is preferably 20, 15, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, Less than 10, 5, 4, 3, 2, or 1 weight percent or molar average by-products are detected. The distance may be less than the boundary.

[0152] The solid composition may be provided in a kit. Such a kit may include a dry container, such as a tube, for example. In some embodiments, the kit comprises two or more wells. Some kits contain multiple dry wells supplied in separate reservoirs. Some kits contain two or more sealed wells in a multi-well plate. The device includes one or more multi-well plates containing a plurality of dry compositions within a well member.

[0153] Some kits also include a reconstitution liquid in a separate vessel from the dry composition. The solid composition may be provided in individual vessels for dispensing aliquots therein, or may be provided in individual vessels for dispensing aliquots therein. or one or more unit dose forms, each combined with a single reservoir containing the solid composition. It is meant to match.

[0154] Optionally, the reservoir containing the solid composition and the reservoir containing the reconstitution liquid may be separated by a frangible material. The fragile materials are aluminum foil, polypropylene, polyester, polyvinyl chloride (P The frangible material may comprise one, two, three or more layers of Each layer may have the same composition or each layer may have a foil in contact with the PVC layer. The films have different compositions, such as layers. , Midland, MI or Arkema, Inc., King of Prussia a, PA. Perforation of the frangible material allows the reconstituted liquid to pass through a solid (e.g., freeze-dried) Allow to come into contact with the composition.

[0155] The kit may be designed to fit into a thermocycler or incubator, Rather, the enzymatic reaction takes place directly within the compartment of the kit, rather than separating the compositions into separate reaction vessels or the like. This avoids the need to transfer the solution to a container that holds the solution.

[0156] The kit may be used to insert a syringe into the reservoir of the kit, for example, by puncturing a port, hose, or septum. Can it be adapted for the introduction of user-supplied reagents into the and US2014 / 0276356), or user-supplied nucleic acid templates, etc. The reagents may be mixed with the reagents of the present disclosure in a user-supplied container. The top is supplied empty and can be used as a mixing chamber.

[0157] All ranges are within their scope, with the understanding that partial numbers refer to the range that the partial numbers refer to. By way of example only, a certain type of oligonucleotide may be used. Nucleotide length ranges for a given sequence are not meaningful because they refer to partial nucleotide units. , does not include partial numbers, and the temperature length range is a partial range because temperature measurements can be partial values. Furthermore, ranges are inclusive of the values ​​that define the range. EXAMPLES

[0158] material and method Multi-well plastic cartridges can be used with commercially available plasma chambers such as the IoN 140 Plasma treatment was performed using corona discharge in air or O2 using a commercially available plasma treatment device. The extent of plasma treatment was monitored by contact angle measurements. The plates typically had contact angles in the range of 75° to 100°. The plasma plates typically exhibited contact angles in the range of 11° to 22°. The treatment reduced the contact angle by approximately 55° to 90°. was a cyclic olefin copolymer.

[0159] An aqueous solution containing trehalose (about 0.15M to about 0.40M) was added to the untreated cartridge. Add approximately 15 to 50 μL per well to the gel wells and plasma-treated cartridge wells. In different experiments, the solutions were dispensed in volumes consistent with the parameters provided above, e.g. The solute concentration of the freeze-dried product was also determined using different cycle lengths. will result in higher throughput unless considerations such as instability favor longer cycles. As mentioned above, shorter cycles are generally preferred in order to provide optimal lyophilization. The parameters depend on the product. The solid composition (pellet) of lyophilized trehalose is The wells of the microplates and cartridges were filled with the eluent.

[0160] Pelletization of untreated and plasma-treated cartridge well surfaces The adhesion of the adhesive was evaluated by performing a drop test as follows.

[0161] Each cartridge to be tested was placed on a sled held at a height of 120 mm on soft foam. The sled was released and allowed to fall onto the foam. The cartridge well was then emptied with the pellets loose. These steps were examined to see whether they were possible. Repeat at successively increasing heights of 60, 440, and 440 mm. Following a fall of 120 mm, the soft foam was replaced with a firm cushion, and the And perform 5 to 8 drops from a height of 440 mm and check that the pellets are not loose after each drop. The results were observed after a 440 mm drop onto a hard cushion followed by a 100 mm drop onto the hard cushion. Replace with a hard surface (concrete or steel) and use the formula h = 20mm x (d-8), where d is Drops 9 to 32 were conducted at height h according to the number of drops (h is the number of drops). For example, drop 9 was 20 m m, fall 10 from 40 mm, etc., and the maximum fall 32 from 480 mm. Drops 33 to 98 were each performed from 480 mm onto a hard surface.

[0162] Example 1. Plasma treatment with short cycle freeze drying 2A-2B show bar graphs in which the height of each bar represents the ratio of the number of treated cartridges ("none") to the number of treated cartridges ("none"). or 12 cartridges of 4 plasma treatment cartridges ("Plasma 1" to "Plasma 4") The number of drops that the pellet withstood in each well before loosening is shown. The experiment was carried out using a

[0163] The adhesion of the pellet to the wells was significantly stronger in the plasma-treated wells. In the well, the pellets became loose after an average of 9.1 drops, whereas in the plasma 1 and plasma For plasma 2, plasma 3, and plasma 4 treated wells, the pellets were an average of 4 It eased off after falls of 6.6, 62.2, 55.7, and 59.4.

[0164] Additional experiments were performed using plasma-treated cartridges in air and O In these experiments, the pellets loosened after an average of 74 to 88 drops (data not shown). not shown).

[0165] Example 2. Plasma treatment with extended cycle freeze drying 3A-3B show bar graphs, with the height of each bar representing an untreated cartridge ("None"); Two cartridges were plasma-treated in air ("Plasma / Air 1" and "Plasma / Air 2”), as well as two cartridges that were plasma-treated in O2 (“Plasma / O Before the pellet loosens in each of the 12 wells ("Plasma / O21" and "Plasma / O22"). The number of drops tolerated is shown. Freeze drying was performed using a longer cycle.

[0166] The adhesion of the pellet to the wells was significantly stronger in the plasma-treated wells. In the well, the pellets became loose after an average of 12.5 drops, whereas in the plasma / air 1, For plasma / air 2, plasma / O2 1, and plasma / O2 2 treated wells, The tethers loosened after an average of 36.9, 48.2, 38.8, and 45.0 falls, respectively. .

[0167] For the plasma 1, plasma 2, plasma 3, and plasma 4 treatments as in Example 1, The provided cartridges were filled with trehalose solution and frozen using a longer cycle. The pellets loosened after an average of 35-41 drops (data not shown). .

[0168] Example 3. Storage of Plasma-treated Cartridges The cartridge was subjected to plasma treatment as in Example 1. After plasma treatment, the cartridge The ridges were stored in an oxygen-containing environment for 1, 30, 90, or 100 days. At the end of the storage period, the wells in the cartridge were filled with a saccharide-containing PCR reaction solution. The solution was freeze-dried. A control cartridge that was not plasma-treated was also loaded with the sugar solution. The samples were filled with 100 ml of 100% ethanol, freeze-dried, and stored for 1, 30, 90, and 100 days. did.

[0169] After lyophilization, all cartridges containing the lyophilized material were stored in a 50-mL aliquot container that mimicked shipping and handling conditions. The packages were subjected to tests designed to mimic the performance of the packages. ) Initial Manual Handling, ASTM D5276-98 Standard, Schedule A, (ii) Loading Pile vibration, ASTM D4728-06 Standard, Schedule D, (iii) Bulk vibration, ASTM D999-08 Standard, Schedule F, (iv) Concentrated Impact, ASTM D63 44-04 Standard, Schedule J, and (v) Final Manual Handling, ASTM D527 After testing, each cartridge containing the lyophilized material was The DNA was visually inspected and used in a PCR assay (reconstituted, combined with a target nucleic acid, amplified and Regardless of storage time, the plasma-treated cartridges were subjected to the same The cartridge contains a dry composition of uniform shape attached to the bottom of the cartridge well, while the untreated cartridge contains a dry composition of uniform shape attached to the bottom of the cartridge well. The P for the material in the plasma treatment cartridge included the disrupted and migrated components. The CR assay performance provided robust and uniform target detection results without any false negatives. PCR assay performance on material in the processing cartridge was inconsistent, resulting in many false negatives. It included.

[0170] Stored in an inert atmosphere (e.g. stored in the presence of oxygen) for at least 100 days ) Plasma-treated cartridges keep lyophilized material intact and fully active after shipping and distribution The cartridges are retained, whereas unprocessed cartridges are not.

[0171] The present disclosure is not limited by the compositions, reagents, methods, diagnostics, laboratory data, etc. of the present disclosure. However, the present disclosure is not limited by any particular or preferred embodiment disclosed herein. Not determined. *******************************

Claims

[Claim 1] A method for preparing a solid composition adhered to a plasma-treated surface, etc.

Images