MAGNETIC IMMUNOSENSOR AND METHOD OF USE

• US 20120034633A1
• Filed: 08/05/2011
• Published: 02/09/2012
• Est. Priority Date: 08/05/2010
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: MILLER, Cary James, Hu, Jinghua
• Granted Patent: US 9,233,370 B2
• Time in Patent Office: Days
• US Class Current: 435/7.94
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• 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 27/745 for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The present invention provides apparatus and methods for the rapid determination of analytes in liquid samples by immunoassays incorporating magnetic capture of beads on a sensor capable of being used in the point-of-care diagnostic field.

Claims

1. A magnetic immunosensing device, comprising:

a sensing electrode on a substantially planar chip, wherein the electrode is positioned in a conduit for receiving a sample mixed with antibody-labeled magnetically susceptible beads; and

an integrated high-field permanent magnetic layer on the chip, wherein the magnetic layer is positioned relative to the electrode, thereby attracting the beads substantially proximate to the electrode and substantially retaining the beads at the electrode surface during removal of unbound sample and washing of the electrode.

2. The device of claim 1, wherein the electrode is microfabricated.

3. The device of claim 1, wherein the magnetic layer is microfabricated.

4. The device of claim 1, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, interstitial fluid and cerebrospinal fluid.

5. The device of claim 1, wherein the sample is amended with an anticoagulant.

6. The device of claim 1, wherein the magnetic layer comprises a neodymium iron boron (NdFeB) alloy.

7. The device of claim 1, wherein the magnetic layer comprises a Nd₂Fe₁₄B alloy.

8. The device of claim 1, wherein the magnetic layer comprises a Nd₂Fe₁₄B alloy in an immobilization matrix.

9. The device of claim 1, wherein the magnetic layer comprises a Nd₂Fe₁₄B alloy in a photoformable polyimide matrix.

10. The device of claim 1, wherein the magnetic layer comprises a Nd₂Fe₁₄B alloy in a polyvinyl alcohol matrix.

11. The device of claim 1, wherein the magnetic layer provides a magnetic field of greater than about 0.1 Tesla.

12. The device of claim 1, wherein the magnetic layer is positioned to yield an event horizon in the conduit in the range of up to about 200 μ

m in the region of the electrode.

13. The device of claim 1, wherein the beads comprise ferrite.

14. The device of claim 1, wherein the beads comprise magnetite (Fe₃O₄).

15. The device of claim 1, wherein the beads comprise a magnetically susceptible core with a polymer coating.

16. The device of claim 1, wherein the beads have an average particle size of from 0.01 μ

m to 20 μ

m.

17. The device of claim 1, wherein the beads have an average particle size of from 0.1 μ

m to 5 μ

m.

18. The device of claim 1, wherein the beads have an average particle size of from 0.2 μ

m to 1.5 μ

m.

19. The device of claim 1, wherein the beads dissolve from a dry matrix into the sample.

20. The device of claim 1, wherein over 50 wt. % of the beads are retained at the electrode surface.

21. The device of claim 1, wherein over 75 wt. % of the beads are retained at the electrode surface.

22. The device of claim 1, wherein the electrode is amperometric.

23. The device of claim 1, wherein the electrode is a gold microarray.

24. The device of claim 1, wherein the conduit has a height of about 0.2 mm to about 5 mm and a width of about 0.2 mm to about 5 mm.

25. The device of claim 1, wherein the conduit has a uniform cross-sectional area.

26. The device of claim 1, wherein the conduit has a non-uniform cross-sectional area.

27. The device of claim 1, wherein the antibody is to a low-abundance analyte in the sample.

28. The device of claim 1, wherein the antibody is to an analyte selected from the group consisting of cardiac troponin I, troponin T, a troponin complex, human chorionic gonadotropin, BNP, creatine kinase, creatine kinase subunit M, creatine kinase subunit B, creatine kinase MB (CK-MB), proBNP, NT-proBNP, myoglobin, myosin light chain and modified fragments thereof.

29. The device of claim 1, wherein the antibody is to an analyte selected from the group consisting of beta-HCG, TSH, ultra hTSH II, TT3, TT4, FT3, FT4, myeloperoxidase, D-dimer, CRP, NGAL, PSA, LH, FSH, prolactin, progesterone, estradiol, DHEA-S, AFP, CA 125 II, CA 125, CA 15-3, CA 19-9, CA 19-9XR, CEA, thyroxine (T4), triiodothyronine (T3), T-uptake, Tg, anti-Tg, anti-TPO, ferritin, cortisol, insulin, HBsAg, HCV Ag/Ab combo, HCV core Ag, anti-HCV, AUSAB (anti-HBs), CORE, CORE-M, SHBG, iPTH, theophylline, sirolimus, tacrolimus, anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M, HAVAB-M2.0, HAVAB-G, HAVAB 2.0, HAVAB 2.0 Quant, IgM, CMV IgM, CMV IgG, â

-2-microglobulin, digitoxin, HBe, anti-HBe, HBeAg, HIV 1/2gO, HIV Ag/Ab combo, testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc, rubella IgG, rubella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgG Avidity, toxo IgG avidity, toxo IgG, toxo IgM, C-peptide, vitamin D, HTLV I/II, total â

hCG, progesterone, estradrogen, prolactin, myoglobin, tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin, NAPA, phenytoin, phenobarbital, valproic acid, vancomycin, procaine, quinidine, tobramycin, methamphetamine (METH), amphetamine (AMPH), barbiturates, benzodiazepine, cannabis, cocaine, methadone, opiates, PCP, acetaminophen, ethanol, salicylates, tricyclics, holoTc, anti-CCP, HbA1c, barbs-U and modified fragments thereof.

30. The device of claim 1, wherein the washing of the electrode is performed with a wash fluid applied as at least one meniscus.

31. A microfabricated magnetic layer on a substantially planar surface, comprising:

high-field permanent magnetic particulates, wherein said particulates are dispersed in a thermally, chemically or photoformably cured immobilization matrix; and

a microfabricated sensing electrode.

32. The microfabricated magnetic layer claim 31, wherein the magnetic particulates comprise a neodymium iron boron (NdFeB) alloy.

33. The microfabricated magnetic layer claim 31, wherein the magnetic particulates comprise a Nd₂Fe₁₄B alloy.

34. The microfabricated magnetic layer claim 31, wherein the magnetic particulates have an average particle size of from 0.01 μ

m to 20 μ

m.

35. The microfabricated magnetic layer claim 31, wherein the magnetic particulates have an average particle size of from 0.01 μ

m to 5 μ

m.

36. The microfabricated magnetic layer claim 31, wherein the immobilization matrix comprises polyimide.

37. The microfabricated magnetic layer claim 31, wherein the immobilization matrix comprises polyvinyl alcohol.

38. The microfabricated magnetic layer claim 31, wherein the magnetic particulates provide a magnetic field of greater than about 0.1 Tesla.

39. The microfabricated magnetic layer claim 31, wherein the magnetic layer is positioned to yield an event horizon for magnetically susceptible beads in the range of less than about 200 μ

m in the region of the electrode.

40. The microfabricated magnetic layer claim 31, wherein the electrode is amperometric.

41. The microfabricated magnetic layer claim 31, wherein the electrode is a gold microarray.

42. A method of performing a sandwich immunoassay for an analyte in a sample with a magnetic immunosensor, wherein said immunosensor comprises a sensing electrode on a substantially planar chip and an integrated layer on said chip that is magnetized and positioned substantially proximate to the electrode, said method comprising:

(a) mixing magnetically susceptible beads coated with a capture antibody to an analyte with a sample containing the analyte and a signal antibody to form a sandwich on said beads;

(b) applying the mixture to the immunosensor;

(c) magnetically localizing and retaining at least a portion of said beads on the electrode;

(d) washing the unbound sample from the electrode;

(e) exposing the signal antibody of the sandwich to a signal generating reagent; and

(f) measuring a signal from the reagent at the electrode.

43. The method of claim 42, wherein the electrode is microfabricated.

44. The method of claim 42, wherein the magnetic layer is microfabricated.

45. The method of claim 42, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, interstitial fluid and cerebrospinal fluid.

46. The method of claim 42, wherein the sample is amended with an anticoagulant.

47. The method of claim 42, wherein the magnetized integrated layer comprises a neodymium iron boron (NdFeB) alloy.

48. The method of claim 42, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy.

49. The method of claim 42, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in an immobilization matrix.

50. The method of claim 42, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in a photoformable polyimide matrix.

51. The method of claim 42, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in a polyvinyl alcohol matrix.

52. The method of claim 42, wherein the magnetized integrated layer provides a magnetic field of greater than about 0.1 Tesla.

53. The method of claim 42, wherein the magnetized integrated layer is positioned to yield an event horizon in the range of up to about 200 μ

m in the region of the electrode.

54. The method of claim 42, wherein the beads comprise ferrite.

55. The method of claim 42, wherein the beads comprise magnetite (Fe₃O₄).

56. The method of claim 42, wherein the beads comprise a magnetically susceptible core with a polymer coating.

57. The method of claim 42, wherein the beads have an average particle size of from 0.01 μ

m to 20 μ

m.

58. The method of claim 42, wherein the beads have an average particle size of from 0.1 μ

m to 5 μ

m.

59. The method of claim 42, wherein the beads have an average particle size of from 0.2 μ

m to 1.5 μ

m.

60. The method of claim 42, wherein the beads dissolve from a dry matrix into the sample.

61. The method of claim 42, wherein the signal antibody dissolves from a dry matrix into the sample.

62. The method of claim 42, wherein over 50 wt. % of the beads are retained at said electrode surface.

63. The method of claim 42, wherein over 75 wt. % of the beads are retained at said electrode surface.

64. The method of claim 42, wherein the electrode is amperometric.

65. The method of claim 42, wherein the electrode is a gold microarray.

66. The method of claim 42, wherein step (a) occurs in a first portion of the immunosensor and step (b) occurs in a second portion of the immunosensor.

67. The method of claim 42, wherein the antibody is to a low-abundance analyte in the sample.

68. The method of claim 42, wherein the antibody is to an analyte selected from the group consisting of cardiac troponin I, troponin T, a troponin complex, human chorionic gonadotropin, BNP, creatine kinase, creatine kinase subunit M, creatine kinase subunit B, creatine kinase MB (CK-MB), proBNP, NT-proBNP, myoglobin, myosin light chain and modified fragments thereof.

69. The method of claim 42, wherein the antibody is to an analyte selected from the group consisting of beta-HCG, TSH, ultra hTSH II, TT3, TT4, FT3, FT4, myeloperoxidase, D-dimer, CRP, NGAL, PSA, LH, FSH, prolactin, progesterone, estradiol, DHEA-S, AFP, CA 125 II, CA 125, CA 15-3, CA 19-9, CA 19-9XR, CEA, thyroxine (T4), triiodothyronine (T3), T-uptake, Tg, anti-Tg, anti-TPO, ferritin, cortisol, insulin, HBsAg, HCV Ag/Ab combo, HCV core Ag, anti-HCV, AUSAB (anti-HBs), CORE, CORE-M, SHBG, iPTH, theophylline, sirolimus, tacrolimus, anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M, HAVAB-M2.0, HAVAB-G, HAVAB 2.0, HAVAB 2.0 Quant, IgM, CMV IgM, CMV IgG, â

-2-microglobulin, digitoxin, HBe, anti-HBe, HBeAg, HIV 1/2gO, HIV Ag/Ab combo, testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc, rubella IgG, rubella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgG Avidity, toxo IgG avidity, toxo IgG, toxo IgM, C-peptide, vitamin D, HTLV I/II, total â

hCG, progesterone, estradrogen, prolactin, myoglobin, tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin, NAPA, phenytoin, phenobarbital, valproic acid, vancomycin, procaine, quinidine, tobramycin, methamphetamine (METH), amphetamine (AMPH), barbiturates, benzodiazepine, cannabis, cocaine, methadone, opiates, PCP, acetaminophen, ethanol, salicylates, tricyclics, holoTc, anti-CCP, HbA1c, barbs-U and modified fragments thereof.

70. The method of claim 42, wherein the washing of said electrode is performed with a wash fluid applied as at least one meniscus.

71. The method of claim 42, performed by a system comprising a reader and a single-use cartridge containing the immunosensor.

72. A method of performing a competitive immunoassay for an analyte in a sample with a magnetic immunosensor, wherein said immunosensor comprises a sensing electrode on a substantially planar chip and an integrated layer on said chip that is magnetized and positioned substantially proximate to said electrode, said method comprising:

(a) mixing magnetically susceptible beads coated with a capture antibody with a sample containing a first analyte and a second analyte, wherein the second analyte is labeled, to permit binding on said beads;

(b) applying the mixture to the immunosensor;

(c) magnetically localizing and retaining at least a portion of the beads on the electrode;

(d) washing the unbound sample from the electrode;

(e) exposing the second analyte to a signal generating reagent; and

(f) measuring a signal from said reagent at the electrode.

73. The method of claim 72, wherein the electrode is microfabricated.

74. The method of claim 72, wherein the magnetic layer is microfabricated.

75. The method of claim 72, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, interstitial fluid and cerebrospinal fluid.

76. The method of claim 72, wherein the sample is amended with an anticoagulant.

77. The method of claim 72, wherein the magnetized integrated layer comprises a neodymium iron boron (NdFeB) alloy.

78. The method of claim 72, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy.

79. The method of claim 72, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in an immobilization matrix.

80. The method of claim 72, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in a photoformable polyimide matrix.

81. The method of claim 72, wherein the magnetized integrated layer comprises a Nd₂Fe₁₄B alloy in a polyvinyl alcohol matrix.

82. The method of claim 72, wherein the magnetized integrated layer provides a magnetic field of greater than about 0.1 Tesla.

83. The method of claim 72, wherein the magnetized integrated layer is positioned to yield an event horizon in the range of up to about 200 μ

m in the region of the electrode.

84. The method of claim 72, wherein the beads comprise ferrite.

85. The method of claim 72, wherein the beads comprise magnetite (Fe₃O₄).

86. The method of claim 72, wherein the beads comprise a magnetically susceptible core with a polymer coating.

87. The method of claim 72, wherein the beads have an average particle size of from 0.01 μ

m to 20 μ

m.

88. The method of claim 72, wherein the beads have an average particle size of from 0.1 μ

m to 5 μ

m.

89. The method of claim 72, wherein the beads have an average particle size of from 0.2 μ

m to 1.5 μ

m.

90. The method of claim 72, wherein the beads dissolve from a dry matrix into the sample.

91. The method of claim 72, wherein the signal antibody dissolves from a dry matrix into the sample.

92. The method of claim 72, wherein the first analyte is in one or more dissolvable dry reagent coatings.

93. The method of claim 72, wherein over 50 wt. % of the beads are retained at said electrode surface.

94. The method of claim 72, wherein over 75 wt. % of the beads are retained at said electrode surface.

95. The method of claim 72, wherein the electrode is amperometric.

96. The method of claim 72, wherein the electrode is a gold microarray.

97. The method of claim 72, wherein step (a) occurs in a first portion of the immunosensor and step (b) occurs in a second portion of the immunosensor.

98. The method of claim 72, wherein the antibody is to a low-abundance analyte in the sample.

99. The method of claim 72, wherein the antibody is to an analyte selected from the group consisting of cardiac troponin I, troponin T, a troponin complex, human chorionic gonadotropin, BNP, creatine kinase, creatine kinase subunit M, creatine kinase subunit B, creatine kinase MB (CK-MB), proBNP, NT-proBNP, myoglobin, myosin light chain and modified fragments thereof.

100. The method of claim 72, wherein the antibody is to an analyte selected from the group consisting of beta-HCG, TSH, ultra hTSH II, TT3, TT4, FT3, FT4, myeloperoxidase, D-dimer, CRP, NGAL, PSA, LH, FSH, prolactin, progesterone, estradiol, DHEA-S, AFP, CA 125 II, CA 125, CA 15-3, CA 19-9, CA 19-9XR, CEA, thyroxine (T4), triiodothyronine (T3), T-uptake, Tg, anti-Tg, anti-TPO, ferritin, cortisol, insulin, HBsAg, HCV Ag/Ab combo, HCV core Ag, anti-HCV, AUSAB (anti-HBs), CORE, CORE-M, SHBG, iPTH, theophylline, sirolimus, tacrolimus, anti-HAV, anti-HAV IgM, HAVAB, HAVAB-M, HAVAB-M2.0, HAVAB-G, HAVAB 2.0, HAVAB 2.0 Quant, IgM, CMV IgM, CMV IgG, â

-2-microglobulin, digitoxin, HBe, anti-HBe, HBeAg, HIV 1/2gO, HIV Ag/Ab combo, testosterone, SCC, vitamin B12, folate, syphilis, anti-HBc, rubella IgG, rubella IgM, homocysteine, MPO, cytomegalovirus (CMV) IgG Avidity, toxo IgG avidity, toxo IgG, toxo IgM, C-peptide, vitamin D, HTLV I/II, total â

hCG, progesterone, estradrogen, prolactin, myoglobin, tPSA, fPSA, carbamazepine (CBZ), digoxin, gentamicin, NAPA, phenytoin, phenobarbital, valproic acid, vancomycin, procaine, quinidine, tobramycin, methamphetamine (METH), amphetamine (AMPH), barbiturates, benzodiazepine, cannabis, cocaine, methadone, opiates, PCP, acetaminophen, ethanol, salicylates, tricyclics, holoTc, anti-CCP, HbA1c, barbs-U and modified fragments thereof.

101. The method of claim 72, wherein the washing of said electrode is performed with a wash fluid applied as at least one meniscus.

102. The method of claim 72, wherein the second analyte is in one or more dissolvable dry reagent coatings.

103. The method of claim 72, wherein the second analyte is labeled with a label selected from the group consisting of a radiolabel, an enzyme, a chromophore, a flurophore, a chemiluminescent species, an ionophore and an electroactive species.

104. The method of claim 72, wherein said labeled analyte is labeled with a label selected from the group consisting of fluorescein, ferrocene and p-aminophenol.

105. The method of claim 72, performed by a system comprising a reader and a single-use cartridge containing the immunosensor.