Polymer brushes for immobilizing molecules to a surface or substrate, where the polymers have water-soluble or water-dispersible segments and probes bonded thereto

• US 6,692,914 B1
• Filed: 07/03/2000
• Issued: 02/17/2004
• Est. Priority Date: 07/02/1999
• Status: Expired due to Fees

• Current Assignee: Freeslate Inc. (Unchained Labs Inc.)
• Original Assignee: Symyx Technologies Incorporated (Dassault Systemes SA)
• Inventors: Charmot, Dominique, Klaerner, Gerrit, Jandeleit, Bernd, Mazzola, Laura T., Benoit, Didier, Nielsen, Ralph B., Mansky, Paul
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/609,461
• Time in Patent Office: 1,324 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 536/22.1, 536/25.3
• US Class Current: 435/6.11
• CPC Class Codes: C08F 220/54 Amides , e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
• CPC Class Codes: C08F 4/00 Polymerisation catalysts
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y10T 428/8305 Miscellaneous [e.g., treated surfaces, etc.]
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

Sensors for determining the presence and concentration of bio-molecules in a biological sample are provided in the form of polymer brushes, which comprise a substrate having a surface that is modified with a water-dispersible or water-soluble polymer segment having functional groups that bind probes. The method of synthesis of such sensors preferably includes use of controlled free radical polymerization techniques, which allows for controlled architecture polymers to modify the surface of the substrate. In this manner functional groups in the polymer chain are removed from the surface, which allows for solution chemistry to be more realistically reproduced with the benefits of a solid bound probe.

Claims

1. A sensor for detecting a biological molecule in an aqueous sample comprising:

(A) a substrate having a surface; and

(B) a plurality of random copolymer chains bound to said surface, each of said plurality of random copolymer chains comprising (i) a water-soluble or water-dispersible segment and (ii) at least one probe bonded to said water-soluble or water-dispersible segment, the probe being selective for the biological molecule, wherein said random copolymer chains are synthesized according to a process comprising;

(i) bonding an initiator to said surface at one or more points to form a derivatized surface;

(ii) contacting said derivatized surface with a composition comprising a water-soluble or water-dispersible free radically polymerizable monomer under reaction conditions to form bound random copolymer chains comprising one or more functional groups on said chains that are available for reaction with the probe; and

,

(iii) bonding said probes to said bound random copolymer chains through said functional groups.

2. A sensor according to claim 1 wherein said derivatized surface is contacted with a composition further comprising an unbound initiator to form both bound and unbound polymer chains, and further wherein said unbound polymer chains are subsequently separated from said bound polymer chains, said unbound initiator comprising an initiator-control agent adduct having the formula:

where Y is a residue capable of initiating a free radical polymerization upon homolytic cleavage of the Y—

O bond, the residue being selected from the group consisting of fragments derived from a free radical initiator, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof;

X is a moiety that is capable of destabilizing the control agent on a polymerization time scale; and

each R¹, R², and R³, independently, is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, heterocycloalkyl, alkoxy, aryloxy, silyl, boryl, phosphino, amino, thio, seleno, and combinations thereof; and

further wherein said bound initiator comprises an initiator-control agent adduct having the formula;

where C is a moiety on the surface of said substrate;

L is a linker group capable of bonding to at least one C moiety;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

z is 0 or 1; and

Y has the same identity as in the unbound initiator, with each R being independently selected from the group consisting of hydrocarbyl and substituted hydrocarbyl.

3. A sensor according to claim 2 wherein X is hydrogen.

4. A sensor according to claim 2 wherein R¹ is selected from the group consisting of hydrogen and methyl.

5. A sensor according to claim 2 wherein C is derived from a hydroxyl group.

6. A sensor according to claim 3 wherein z=1, q=1, r=1, and t=0.

7. A sensor according to claim 4 wherein z=1, q=1, r=1, and t=1.

8. A sensor according to claim 1 further comprising bonding spacer molecules to said substrate surface at one or more points different from the points at which said polymer chains are bound, to space said bond polymer chains apart from each other.

9. A sensor according to claim 8 wherein said spacer molecules bound to said surface are characterized by the formula:

where C is a moiety on the surface of said substrate;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

L is a linker group capable of bonding to at least one C moiety;

z is 0 or 1; and

Q is a group incapable of initiating free radical polymerization that is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof.

10. A sensor according to claim 1 wherein said functional groups are selected from the group consisting of hydroxyl, amino, carboxyl, and combinations thereof.

22. The sensor of claim 1 wherein the polymer chains have a weight average molecular weight of at least 50,000.

23. The sensor of claim 1 wherein the polymer chains have a weight average molecular weight of at least 100,000.

24. The sensor of claim 1 wherein the polymer chains have a weight average molecular weight of at least 500,000.

25. The sensor of claim 1 wherein said polymer chains are linear.

26. The sensor of claim 1 wherein said polymer chains are non-linear.

27. The sensor of claim 26 wherein said non-linear polymers are selected from the group consisting of branched polymers, star polymers, and combinations thereof.

28. The sensor of claim 1 wherein said polymer chains comprise units having the formula:

wherein R⁴ is H or an alkyl group; and

R⁵ and R⁶, independently, are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, alkoxy, aryloxy, and combinations thereof.

29. The sensor of claim 1 wherein said polymer chains form a layer on said substrate surface having a dry thickness ranging from about 20 angstroms to about 1000 angstroms.

30. The sensor of claim 1 wherein said substrate is a bead or pellet.

31. The sensor of claim 1 wherein the surface of said substrate is gold.

32. The sensor of claim 8 wherein a ratio of polymer chains to a sum of polymer chains and spacer molecules is less than about 0.75:

1.

11. A method of preparing a sensor for detecting a biological molecule in an aqueous sample, the method comprising:

(i) bonding an initiator to a surface of a substrate at one or more points to form a derivatized surface;

(ii) contacting said derivatized surface with a composition comprising a water-soluble or water-dispersible free radically polymerizable monomer under reaction conditions to form bound random copolymer chains comprising;

(a) a water-soluble or water-dispersible segment having a weight average molecular weight of at least about 1,000; and

(b) one or more functional groups on the random copolymer chains that are capable of reacting with a probe selective for the biological molecule; and

,

(iii) bonding the probe to said bound random copolymer chains through said functional groups.

12. A method according to claim 11 wherein said derivatized surface is contacted with a composition further comprising an unbound initiator to form both bound and unbound polymer chains, said unbound polymer chains being subsequently separated from said bound polymer chains, and said unbound initiator comprising an initiator-control agent adduct having the formula:

where Y is a residue capable of initiating a free radical polymerization upon homolytic cleavage of the Y—

O bond, the residue being selected from the group consisting of fragments derived from a free radical initiator, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof;

X is a moiety that is capable of destabilizing the control agent on a polymerization time scale; and

each R¹, R², and R³, independently, is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, heterocycloalkyl, alkoxy, aryloxy, silyl, boryl, phosphino, amino, thio, seleno, and combinations thereof; and

further wherein said bound initiator comprises an initiator-control agent adduct having the formula;

where C is a moiety on the surface of said substrate;

L is a linker group capable of bonding to at least one C moiety;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

z is 0 or 1; and

Y, R¹, R², R³, and X have the same identity as in the case of the unbound initiator.

13. A method according to claim 12 wherein X is hydrogen.

14. A method according to claim 12 wherein R¹ is selected from the group consisting of hydrogen and methyl.

15. A method according to claim 12 wherein C is a hydroxyl group.

16. A method according to claim 12 wherein z=1, q=1, r=1, and t=0.

17. A method according to claim 12 wherein z=1, q=1, r=1, and t=1.

18. A method according to claim 11 further comprising bonding spacer molecules to said substrate surface at one or more points different from the points at which said polymer chains are bound, to space said bound polymer chains apart from each other.

19. A method according to claim 18 wherein said spacer molecules bound to said surface are characterized by the formula:

where C is a moiety on the surface of said substrate;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

L is a linker group capable of bonding to at least one C moiety;

z is 0 or 1; and

Q is a group incapable of initiating free radical polymerization that is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof.

20. A method according to claim 11 wherein said functional groups are selected from the group consisting of hydroxyl, amino, carboxyl, and combinations thereof.

21. The method of claim 20 wherein the functional groups are activated with phosgene, thiophosgene, 4-methyl-phenylsulfonylchoride, methylsulfonylchloride, a carbodiimide, periodate, a bis-aldehyde or a di-amino composition.

33. The method of claim 11 wherein the polymer chains have a weight average molecular weight of at least 50,000.

34. The method of claim 11 wherein the polymer chains have a weight average molecular weight of at least 100,000.

35. The method of claim 11 wherein the polymer chains have a weight average molecular weight of at least 500,000.

36. The method of claim 11 wherein said polymer chains are linear.

37. The method of claim 11 wherein said polymer chains are non-linear.

38. The method of claim 37 wherein said non-linear polymers are selected from the group consisting of branched polymers, star polymers, and combinations thereof.

39. The method of claim 11 wherein said polymer chains comprise units having the formula:

wherein R⁴ is H or an alkyl group; and

R⁵ and R⁶, independently, are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, alkoxy, aryloxy, and combinations thereof.

40. The method of claim 11 wherein said polymer chains form a layer on said substrate surface having a dry thickness ranging from about 20 angstroms to about 1000 angstroms.

41. The method of claim 11 wherein said substrate is a bead or pellet.

42. The method of claim 11 wherein the surface of said substrate is gold.

43. The method of claim 18 wherein a ratio of polymer chains to a sum of polymer chains and spacer molecules is less than about 0.75:

1.

44. A sensor for detecting a biological molecule in an aqueous sample comprising:

(A) a substrate having a surface; and

(B) a plurality of polymer chains bound to said surface, each of said plurality of polymer chains comprising (i) a water-soluble or water-dispersible segment and (ii) at least one probe bonded to said water-soluble or water-dispersible segment, the probe being selective for the biological molecule, wherein said polymer chains are synthesized according to a process comprising;

(i) bonding an initiator to said surface at one or more points to form a derivatized surface;

(ii) contacting said derivatized surface with a composition comprising a water-soluble or water-dispersible free radically polymerizable monomer under reaction conditions to form bound polymer chains comprising one or more functional groups on said chains that are available for reaction with the probe, said water-soluble or water-dispersible monomer having the formula;

wherein R⁴ is H or an alkyl group; and

R⁵ and R⁶, independently, are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, alkoxy, aryloxy, and combinations thereof; and

,

(iii) bonding said probes to said bound polymer chains through said functional groups.

45. A sensor according to claim 44 wherein said derivatized surface is contacted with a composition further comprising an unbound initiator to form both bound and unbound polymer chains, and further wherein said unbound polymer chains are subsequently separated from said bound polymer chains, said unbound initiator comprising an initiator-control agent adduct having the formula:

where Y is a residue capable of initiating a free radical polymerization upon homolytic cleavage of the Y—

O bond, the residue being selected from the group consisting of fragments derived from a free radical initiator, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof;

X is a moiety that is capable of destabilizing the control agent on a polymerization time scale; and

each R¹, R², and R³, independently, is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, heterocycloalkyl, alkoxy, aryloxy, silyl, boryl, phosphino, amino, thio, seleno, and combinations thereof; and

further wherein said bound initiator comprises an initiator-control agent adduct having the formula;

where C is a moiety on the surface of said substrate;

L is a linker group capable of bonding to at least one C moiety;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

z is 0 or 1; and

Y has the same identity as in the unbound initiator, with each R being independently selected from the group consisting of hydrocarbyl and substituted hydrocarbyl.

46. A sensor according to claim 45 wherein X is hydrogen.

47. A sensor according to claim 46 wherein z=1, q=1, r=1, and t=0.

48. A sensor according to claim 45 wherein R¹ is selected from the group consisting of hydrogen and methyl.

49. A sensor according to claim 48 wherein z=1, q=1, r=1, and t=1.

50. A sensor according to claim 45 wherein C is derived from a hydroxyl group.

51. A sensor according to claim 44 further comprising bonding spacer molecules to said substrate surface at one or more points different from the points at which said polymer chains are bound, to space said bound polymer chains apart from each other.

52. A sensor according to claim 51 wherein said spacer molecules bound to said surface are characterized by the formula:

where C is a moiety on the surface of said substrate;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

L is a linker group capable of bonding to at least one C moiety;

z is 0 or 1; and

Q is a group incapable of initiating free radical polymerization that is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof.

53. A sensor according to claim 44 wherein said functional groups are selected from the group consisting of hydroxyl, amino, carboxyl, and combinations thereof.

54. A sensor according to claim 44 wherein the polymer chains have a weight average molecular weight of at least 100,000.

55. A sensor according to claim 44 wherein the polymer chains have a weight average molecular weight of at least 500,000.

56. A sensor according to claim 44 wherein said polymer chains are linear.

57. A sensor according to claim 44 wherein said polymer chains are non-linear.

58. A sensor according to claim 44 wherein said polymer chains are homopolymers.

59. A sensor according to claim 44 wherein said polymer chains are copolymers.

60. A sensor according to claim 44 wherein said polymer chains form a layer on said substrate surface having a dry thickness ranging from about 20 angstroms to about 1000 angstroms.

61. A sensor according to claim 44 wherein said substrate is a bead or pellet.

62. A sensor according to claim 44 wherein the surface of said substrate is gold.

63. A method of preparing a sensor for detecting a biological molecule in an aqueous sample, the method comprising:

(i) bonding an initiator to a surface of a substrate at one or more points to form a derivatized surface;

(ii) contacting said derivatized surface with a composition comprising a water-soluble or water-dispersible free radically polymerizable monomer under reaction conditions to form bound polymer chains comprising;

(a) a water-soluble or water-dispersible segment having a weight average molecular weight of at least about 1,000; and

(b) one or more functional groups on the polymer chains that are capable of reacting with a probe selective for the biological molecule, said water-soluble or water-dispersible monomer having the formula;

wherein R⁴ is H or an alkyl group; and

R⁵ and R⁶, independently, are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, alkoxy, aryloxy, and combinations thereof; and

,

(iii) bonding the probe to said bound polymer chains through said functional groups.

64. A method according to claim 63 wherein said derivatized surface is contacted with a composition further comprising an unbound initiator to form both bound and unbound polymer chains, said unbound polymer chains being subsequently separated from said bound polymer chains, and said unbound initiator comprising an initiator-control agent adduct having the formula:

where Y is a residue capable of initiating a free radical polymerization upon homolytic cleavage of the Y—

O bond, the residue being selected from the group consisting of fragments derived from a free radical initiator, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof;

X is a moiety that is capable of destabilizing the control agent on a polymerization time scale; and

each R¹, R², and R³, independently, is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, heterocycloalkyl, alkoxy, aryloxy, silyl, boryl, phosphino, amino, thio, seleno, and combinations thereof; and

further wherein said bound initiator comprises an initiator-control agent adduct having the formula;

where C is a moiety on the surface of said substrate;

L is a linker group capable of bonding to at least one C moiety;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

z is 0 or 1; and

Y has the same identity as in the unbound initiator, with each R being independently selected from the group consisting of hydrocarbyl and substituted hydrocarbyl.

65. A method according to claim 64 wherein X is hydrogen.

66. A method according to claim 65 wherein z=1, q=1, r=1, and t=0.

67. A method according to claim 64 wherein R¹ is selected from the group consisting of hydrogen and methyl.

68. A method according to claim 67 wherein z=1, q=1, r=1, and t=1.

69. A method according to claim 64 wherein C is derived from a hydroxyl group.

70. A method according to claim 63 further comprising bonding spacer molecules to said substrate surface at one or more points different from the points at which said polymer chains are bound, to space said bound polymer chains apart from each other.

71. A method according to claim 70 wherein said spacer molecules bound to said surface are characterized by the formula:

where C is a moiety on the surface of said substrate;

q, r, and t, independently, are 0 or 1, provided at least one of q, r and t is 1;

L is a linker group capable of bonding to at least one C moiety;

z is 0 or 1; and

Q is a group incapable of initiating free radical polymerization that is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, and combinations thereof.

72. A method according to claim 63 wherein said functional groups are selected from the group consisting of hydroxyl, amino, carboxyl, and combinations thereof.

73. A method according to claim 63 wherein the polymer chains have a weight average molecular weight of at least 100,000.

74. A method according to claim 63 wherein the polymer chains have a weight average molecular weight of at least 500,000.

75. A method according to claim 63 wherein said polymer chains are linear.

76. A method according to claim 63 wherein said polymer chains are non-linear.

77. A method according to claim 63 wherein said polymer chains are homopolymers.

78. A method according to claim 63 wherein said polymer chains are copolymers.

79. A method according to claim 63 wherein said polymer chains form a layer on said substrate surface having a dry thickness ranging from about 20 angstroms to about 1000 angstroms.

80. A method according to claim 63 wherein said substrate is a bead or pellet.

81. A method according to claim 63 wherein the surface of said substrate is gold.