Sensors for assaying coagulation in fluid samples

• US 9,903,877 B2
• Filed: 09/25/2015
• Issued: 02/27/2018
• Est. Priority Date: 09/26/2014
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Inventors: Zhao, Tian-Xian, Wong, Anjulia Chiu-Yuk, Martin, Glenn Brian, Di Tullio, Katrina Petronilla, Thibodeau, Isabelle Louisette, Breeze, Steven Roy, Sutrala, Smitha R K, Hong, Eric, Ball, Sheila Diane
• Primary Examiner(s): Cordero Garcia, Marcela M
• Application Number:
  US14/866,266
• Publication Number:
  US 20160091507A1
• Time in Patent Office: 886 Days
• CPC Class Codes: B01L 2200/04 Exchange or ejection of cartridges, containers or reservoirs
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/024 Storing results with means integrated into the container
• CPC Class Codes: B01L 2300/027 Digital display, e.g. LCD, LED
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0472 Diffusion
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 2333/96463 : Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 33/86 : involving blood coagulating time or factors, or their receptors

Abstract

The present invention relates to analytical testing devices including micro-environment sensors and methods for assaying coagulation in a fluid sample applied to the micro-environment sensors, and in particular, performing one or more types of coagulation assays using one or more micro-environment sensors in a single point of care combined test cartridge. For example, the present invention may be directed to test sensor including at least one transducer coated with a polymer layer. The polymer layer comprises a thrombin-cleavable peptide with a detectable moiety.

Claims

1. A test sensor for a blood coagulation assay comprising at least one transducer coated with a polymer layer, wherein the polymer layer is a porous support layer that comprises immobilized therein a thrombin-cleavable peptide, and wherein the thrombin-cleavable peptide comprises a detectable moiety linked by a thrombin-cleavable amide bond to a polypeptide sequence that is non-reactive with blood proteases other than thrombin.

2. The test sensor of claim 1, wherein the test sensor is a prothrombin time (PT) sensor.

3. The test sensor of claim 2, wherein the polymer layer comprises a heparin-neutralizing polymer.

4. The test sensor of claim 2, wherein the polymer layer comprises a non-heparin-neutralizing polymer.

5. The test sensor of claim 4, wherein the polymer layer is modified to impart a heparin neutralizing property upon the non-heparin-neutralizing polymer.

6. The test sensor of claim 5, wherein the polymer layer is modified to preferentially bind sulfate groups on heparin.

7. The test sensor of claim 2, further comprising a PT reagent formed as a layer over the polymer layer.

8. The test sensor of claim 7, wherein the PT reagent comprises one or more components selected from the group consisting of:

non-recombinant tissue factor, recombinant tissue factor, a natural or synthetic lipid, a natural or synthetic phospholipid, a combination of natural and synthetic lipids, a combination of natural and synthetic phospholipids, protein carrier, a stabilizing agent, an antimicrobial agent, a calcium salt, a potassium salt, a water soluble polymer, a sugar, gelatin, agarose, a polysaccharide, a saccharide, sucrose, polyethylene glycol, sodium phosphate, glycine, an amino acid, antioxidants, a detergent, a buffer salt, and a buffer.

9. The test sensor of claim 2, wherein the polymer layer comprises a PT reagent.

10. The test sensor of claim 9, wherein the PT reagent comprises one or more components selected from the group consisting of:

non-recombinant tissue factor, recombinant tissue factor, a natural or synthetic lipid, a natural or synthetic phospholipid, a combination of natural and synthetic lipids, a combination of natural and synthetic phospholipids, protein carrier, a stabilizing agent, an antimicrobial agent, a calcium salt, a potassium salt, a water soluble polymer, a sugar, gelatin, agarose, a polysaccharide, a saccharide, sucrose, polyethylene glycol, sodium phosphate, glycine, an amino acid, antioxidants, a detergent, a buffer salt, and a buffer.

11. The test sensor of claim 2, further comprising a PT reagent located adjacent to the at least one transducer.

12. The test sensor of claim 11, wherein the PT reagent comprises one or more components selected from the group consisting of:

non-recombinant tissue factor, recombinant tissue factor, a natural or synthetic lipid, a natural or synthetic phospholipid, a combination of natural and synthetic lipids, a combination of natural and synthetic phospholipids, protein carrier, a stabilizing agent, an antimicrobial agent, a calcium salt, a potassium salt, a water soluble polymer, a sugar, gelatin, agarose, a polysaccharide, a saccharide, sucrose, polyethylene glycol, sodium phosphate, glycine, an amino acid, antioxidants, a detergent, a buffer salt, and a buffer.

13. The test sensor of claim 2, wherein the at least one transducer is an electrode comprising a metal selected from the group consisting of:

gold, platinum, palladium, iridium, and alloys thereof.

14. The test sensor of claim 2, wherein the at least one transducer is an optical detector.

15. The test sensor of claim 14, wherein the optical detector is either a detector of fluorescence, chemiluminescence, or bioluminescence emission from the detectable moiety or a detector of absorbance by the detectable moiety.

16. The test sensor of claim 2, wherein the detectable moiety is an electroactive species, an optical dye, or one of a fluorescence, bioluminescence, and chemiluminescence emitter.

17. The test sensor of claim 2, wherein the polymer layer comprises polyvinyl alcohol (PVA).

18. The test sensor of claim 17, wherein the PVA is cross-linked PVA.

19. The test sensor of claim 17, wherein the PVA is a photo-activated stilbizonium salt.

20. The test sensor of claim 1, wherein the polymer layer is composed of one or more of the polymers selected from the group consisting of:

polyvinyl alcohol (PVA), styrylpyridinium polyvinylalcohol (SBQ-PVA), agarose, polyacrylamide, polymethyl methacrylate, N-methylpyrrolidone, polyvinylpyrrolidone, polyimide, a film-forming latex, sepharose, polyurethanes, acrylates, methacrylates, polyethylene glycols, polylactic acid, poly(lactic co-glycolic acid), celluloses, inulin, fructans, polyglycolic acid, and copolymers of vinyl acetate and ethylene, terpolymers of vinyl acetate, ethylene and other comonomers.

21. The test sensor of claim 1, wherein the thrombin-cleavable peptide is selected from the group consisting of:

H-D-Phe-Pip-Arg, H-D-Chg-Abu-Arg, CBZ-Gly-Pro-Arg, Boc-Val-Pro-Arg, H-D-Phe-Pro-Arg, Cyclohexylglycine-Ala-Arg, Tos-Gly-Pro-Arg, Bz-Phe-Val-Arg, Boc-Val-Pro-Arg, Ac-Val-Pro-Arg, Ac-Val-Hyp-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Val-Pro-Arg, Ac-Gly-Pro-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Gly-Pro-Arg, Ac-Gly-Hyp-Arg and H-D-Chg-Abu-Arg.

22. The test sensor of claim 1, wherein the detectable moiety is selected from the group consisting of:

p-aminophenol, a quinone, an organometallic species, p-nitroaniline, o-dianisidine, 4,4′

-benzidine, 4-methoxy-2-naphthylamine, N-phenyl-p-phenylenediamine, N-[p-methoxyphenyl-]-p-phenylenediamine, and phenazine derivatives.

23. The test sensor of claim 1, wherein the test sensor is an activated partial thromboplastin time (aPTT) sensor.

24. The test sensor of claim 23, wherein the polymer layer comprises a non-heparin-neutralizing polymer.

25. The test sensor of claim 23, further comprising aPTT reagent formed as a layer over the polymer layer.

26. The test sensor of claim 25, wherein the aPTT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

27. The test sensor of claim 23, wherein the polymer layer comprises aPTT reagent.

28. The test sensor of claim 27, wherein the aPTT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

29. The test sensor of claim 23, further comprising aPTT reagent located adjacent to the at least one transducer.

30. The test sensor of claim 29, wherein the aPTT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

31. The test sensor of claim 23, wherein the at least one transducer is an electrode comprising a metal selected from the group consisting of:

gold, platinum, palladium, iridium, and alloys thereof.

32. The test sensor of claim 23, wherein the at least one transducer is an optical detector.

33. The test sensor of claim 32, wherein the optical detector is either a detector of fluorescence, chemiluminescence, or bioluminescence emission from the detectable moiety or a detector of absorbance by the detectable moiety.

34. The test sensor of claim 23, wherein the detectable moiety is an electroactive species, an optical dye, or one of a fluorescence, bioluminescence, and chemiluminescence emitter.

35. The test sensor of claim 23, wherein the polymer layer is composed of one or more of the polymers selected from the group consisting of:

copolymers of vinyl acetate and ethylene, terpolymers of vinyl acetate, ethylene and other comonomers, polylactic acid, poly(lactic co-glycolic acid), celluloses, inulin, fructose, fructans, and polyglycolic acid.

36. The test sensor of claim 23, wherein the thrombin-cleavable peptide is selected from the group consisting of:

H-D-Phe-Pip-Arg, H-D-Chg-Abu-Arg, CBZ-Gly-Pro-Arg, Boc-Val-Pro-Arg, H-D-Phe-Pro-Arg, Cyclohexylglycine-Ala-Arg, Tos-Gly-Pro-Arg, Bz-Phe-Val-Arg, Boc-Val-Pro-Arg, Ac-Val-Pro-Arg, Ac-Val-Hyp-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Val-Pro-Arg, Ac-Gly-Pro-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Gly-Pro-Arg, Ac-Gly-Hyp-Arg and H-D-Chg-Abu-Arg.

37. The test sensor of claim 23, wherein the detectable moiety is selected from the group consisting of:

p-aminophenol, a quinone, an organometallic species, p-nitroaniline, o-dianisidine, 4,4′

-benzidine, 4-methoxy-2-naphthylamine, N-phenyl-p-phenylenediamine, N-[p-methoxyphenyl-]-p-phenylenediamine, and phenazine derivatives.

38. The test sensor of claim 1, wherein the test sensor is an activated clotting time (ACT) sensor.

39. The test sensor of claim 38, wherein the polymer layer comprises a non-heparin-neutralizing polymer.

40. The test sensor of claim 38, further comprising an ACT reagent formed as a layer over the polymer layer.

41. The test sensor of claim 40, wherein the ACT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

42. The test sensor of claim 38, wherein the polymer layer comprises an ACT reagent.

43. The test sensor of claim 42, wherein the ACT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

44. The test sensor of claim 38, further comprising an ACT reagent located adjacent to the at least one transducer.

45. The test sensor of claim 44, wherein the ACT reagent comprises one or more components selected from the group consisting of:

ellagic acid, celite, kaolin, diatomaceous earth, clay, silicon dioxide, synthetic or natural lipids, synthetic or natural phospholipids, dextran, dextrin, tergitol, a buffer, a carrier protein, an amino acid, a stabilizer, an antimicrobial, an antioxidant, a detergent, a saccharide, a polysaccharide, sucrose, polyethylene glycol, derivatives of polyethylene glycol, glycine, gelatin, rhamnose, trehalose, and a sugar.

46. The test sensor of claim 38, wherein the at least one transducer is an electrode comprising a metal selected from the group consisting of:

gold, platinum, palladium, iridium, and alloys thereof.

47. The test sensor of claim 38, wherein the at least one transducer is an optical detector.

48. The test sensor of claim 47, wherein the optical detector is either a detector of fluorescence, chemiluminescence, or bioluminescence emission from the detectable moiety or a detector of absorbance by the detectable moiety.

49. The test sensor of claim 38, wherein the detectable moiety is an electroactive species, an optical dye, or one of a fluorescence, bioluminescence, and chemiluminescence emitter.

50. The test sensor of claim 38, wherein the polymer layer is composed of one or more of the polymers selected from the group consisting of:

copolymers of vinyl acetate and ethylene, terpolymers of vinyl acetate, ethylene and other comonomers, polylactic acid, poly(lactic co-glycolic acid), celluloses, inulin, fructose, fructans, and polyglycolic acid.

51. The test sensor of claim 38, wherein the thrombin-cleavable peptide is selected from the group consisting of:

H-D-Phe-Pip-Arg, H-D-Chg-Abu-Arg, CBZ-Gly-Pro-Arg, Boc-Val-Pro-Arg, H-D-Phe-Pro-Arg, Cyclohexylglycine-Ala-Arg, Tos-Gly-Pro-Arg, Bz-Phe-Val-Arg, Boc-Val-Pro-Arg, Ac-Val-Pro-Arg, Ac-Val-Hyp-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Val-Pro-Arg, Ac-Gly-Pro-Arg, Ac-(8-amino-3,6,dioxaoctanoyl-Gly-Pro-Arg, Ac-Gly-Hyp-Arg and H-D-Chg-Abu-Arg.

52. The test sensor of claim 38, wherein the detectable moiety is selected from the group consisting of:

p-aminophenol, a quinone, an organometallic species, p-nitroaniline, o-dianisidine, 4,4′

-benzidine, 4-methoxy-2-naphthylamine, N-phenyl-p-phenylenediamine, N-[p-methoxyphenyl-]-p-phenylenediamine, and phenazine derivatives.

53. The test sensor of claim 1, wherein the polymer layer is a substantially heparin-neutralizing polymer layer comprising cross-linked polyvinyl alcohol (PVA).

54. The test sensor of claim 1, wherein the polymer layer is a substantially non-heparin-neutralizing polymer layer comprising hydroxypropyl cellulose.

57. The test sensor of claim 1, further comprising a reagent for conducting the blood coagulation assay, wherein the reagent is one or more of:

formed as a layer over the polymer layer;

immobilized within the polymer layer; and

is adjacent to the at least one traducer.

58. The test sensor of claim 1, wherein the polymer layer comprises one or more celluloses selected from the group consisting of hydroxypropyl cellulose, hydroxypropylmethylcellulose acetate succinate, and carboxymethyl cellulose.

59. The test sensor of claim 23, wherein the polymer layer comprises one or more celluloses selected from the group consisting of hydroxypropyl cellulose, hydroxypropylmethylcellulose acetate succinate, and carboxymethyl cellulose.

60. The test sensor of claim 38, wherein the polymer layer comprises one or more celluloses selected from the group consisting of hydroxypropyl cellulose, hydroxypropylmethylcellulose acetate succinate, and carboxymethyl cellulose.

61. The test sensor of claim 22, wherein the organometallic species is selected from the group consisting of a ferrocene and a ferrocyanide.

62. The test sensor of claim 37, wherein the organometallic species is selected from the group consisting of a ferrocene and a ferrocyanide.

63. The test sensor of claim 53, wherein the organometallic species is selected from the group consisting of a ferrocene and a ferrocyanide.

55. A method for performing multiple diagnostic clotting time tests on a whole blood sample, the method comprising:

providing a point-of-care test device with two or more test sensors for performing the diagnostic clotting time tests, each of the two or more test sensors comprising at least one transducer coated with a polymer layer, wherein the polymer layer is a porous support layer that comprises immobilized therein a thrombin-cleavable peptide, wherein the thrombin-cleavable peptide comprises a detectable moiety linked by a thrombin-cleavable amide bond to a polypeptide sequence that is non-reactive with blood proteases other than thrombin, and wherein the two or more test sensors comprise a micro-environment prothrombin time (PT) sensor disposed in a first region of a conduit within the test device and a micro-environment activated partial thromboplastin time (aPTT) sensor in a second region of the conduit;

amending the whole blood sample with a PT reagent in the first region and an aPTT reagent in the second region;

waiting a predetermined amount of time for the amended whole blood sample to diffuse into a first thrombin-cleavable peptide, which is an immobilized PT substrate, in the first region and a second thrombin-cleavable peptide, which is an immobilized aPTT substrate, in the second region; and

determining a PT using the PT sensor, and an aPTT using the aPTT sensor.

56. A method for performing multiple diagnostic clotting time tests on a whole blood sample, the method comprising:

providing a point-of-care test device with two or more test sensors for performing the diagnostic clotting time tests, each of the two or more test sensors comprising at least one transducer coated with a polymer layer, wherein the polymer layer is a porous support layer that comprises immobilized therein a thrombin-cleavable peptide, wherein the thrombin-cleavable peptide comprises a detectable moiety linked by a thrombin-cleavable amide bond to a polypeptide sequence that is non-reactive with blood proteases other than thrombin, and wherein the two or more test sensors comprise a micro-environment prothrombin time (PT) sensor disposed in a first region of a conduit within the test device and a micro-environment activated clotting time (ACT) sensor in a second region of the conduit;

amending the whole blood sample with a PT reagent in the first region and an ACT reagent in the second region;

waiting a predetermined amount of time for the amended whole blood sample to diffuse into a first thrombin-cleavable peptide, which is an immobilized PT substrate, in the first region and a second thrombin-cleavable peptide, which is an immobilized ACT substrate, in the second region; and

determining a PT using the PT sensor, and an ACT using the ACT sensor.