U.S. patent application number 09/821396 was filed with the patent office on 2002-05-02 for detection and amplification of ligands.
Invention is credited to Doane, Kathleen J., Ishikawa, Tomohiro, Lavrentovich, Oleg D., Niehaus, Gary D., Woolverton, Christopher J..
Application Number | 20020052002 09/821396 |
Document ID | / |
Family ID | 25233286 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052002 |
Kind Code |
A1 |
Niehaus, Gary D. ; et
al. |
May 2, 2002 |
Detection and amplification of ligands
Abstract
Devices and systems for the detection of ligands comprising at
least one receptor and an amplification mechanism comprising a
liquid crystalline, where an amplified signal is produced as a
result of receptor binding to a ligand are provided. Also provided
are methods for the automatic detection of ligands.
Inventors: |
Niehaus, Gary D.; (Kent,
OH) ; Woolverton, Christopher J.; (Kent, OH) ;
Lavrentovich, Oleg D.; (Kent, OH) ; Ishikawa,
Tomohiro; (Cleveland, OH) ; Doane, Kathleen J.;
(Ravenna, OH) |
Correspondence
Address: |
RENNER, KENNER, GREIVE, BOBAK, TAYLOR & WEBER
FOURTH FLOOR
FIRST NATIONAL TOWER
AKRON
OH
44308
US
|
Family ID: |
25233286 |
Appl. No.: |
09/821396 |
Filed: |
March 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09821396 |
Mar 29, 2001 |
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09633327 |
Aug 7, 2000 |
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09633327 |
Aug 7, 2000 |
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09095196 |
Jun 10, 1998 |
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6171802 |
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Current U.S.
Class: |
435/7.1 ; 349/56;
435/287.2 |
Current CPC
Class: |
G01N 33/551 20130101;
G01N 33/552 20130101; G01N 33/54373 20130101; G01N 33/545
20130101 |
Class at
Publication: |
435/7.1 ;
435/287.2; 349/56 |
International
Class: |
G01N 033/53; C12M
001/34; G02F 001/1333 |
Claims
We claim:
1. A device for the detection of ligands comprising: at least one
substantially spherical substrate; at least one receptor attached
to said spherical substrate, wherein said at least one receptor is
capable of binding to a ligand to form a receptor-ligand complex
and wherein the formation of said receptor-ligand complex produces
a signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
amplifies said signal upon receptor-ligand complex formation.
2. The device of claim 1, wherein said substantially spherical
substrate is non-porous.
3. The device of claim 2, wherein said at least one receptor is
attached to the surface of said non-porous substantially spherical
substrate.
4. The device of claim 1, wherein the substantially spherical
substrate is porous.
5. The device of claim 4, wherein said at least one receptor is
attached to at least one of (i) the surface of said porous
substantially spherical substrate and (ii) the pores of said porous
substantially spherical substrate.
6. The device of claim 5, wherein said at least one receptor is
attached to the surface of said porous substantially spherical
substrate.
7. The device of claim 5, wherein said at least one receptor is
attached to the pores of said porous substantially spherical
substrate.
8. The device of claim 5, wherein a plurality of receptors are
attached to and randomly distributed on the surface and within the
pores of said porous substantially spherical substrate.
9. The device of claim 1, wherein the liquid crystalline material
is selected from the group consisting of thermotropic liquid
crystalline material and lyotropic liquid crystalline material.
10. The device of claim 9, wherein the liquid crystalline material
is a lyotropic liquid crystalline material.
11. The device of claim 10, wherein the lyotropic liquid
crystalline material is a lyotropic chromonic liquid crystalline
material.
12. The device of claim 9, wherein the liquid crystalline material
is a thermotropic liquid crystalline material.
13. The device of claim 1, wherein the substantially spherical
substrate is made from a material selected from the group
consisting of polymeric and inorganic materials.
14. The device of claim 13, wherein the substantially spherical
substrate is made from a polymeric material.
15. The device of claim 14, wherein the polymeric materials are
selected from the group consisting of polyions, polyalkenes,
polyacrylates, polymethacrylates, polyvinyls, polystyrenes,
polycarbonates, polyesters, polyurethanes, polyamides, polyimides,
polysulfones, polysiloxanes, polysilanes, polyethers, and
polycarboxylates.
16. The device of claim 15, wherein the polymeric material is a
polystyrene.
17. The device of claim 13, wherein the substantially spherical
substrate is made from an inorganic material.
18. The device of claim 17, wherein the inorganic material is
selected from the group consisting of glass, silicon, and colloidal
gold.
19. The device of claim 18, wherein the inorganic material is
glass.
20. The device of claim 1, wherein said at least one receptor is
attached to said spherical substrate by one means selected from the
group consisting of (i) chemical attachment and (ii) physical
attachment.
21. The device of claim 20, wherein said at least one receptor is
attached to said spherical substrate by chemical attachment.
22. The device of claim 21, wherein said chemical attachment is
covalent bonding.
23. The device of claim 20, wherein said at least one receptor is
attached to said spherical substrate by physical attachment.
24. The method of claim 23, wherein said physical attachment is
selected from the group consisting hydrophobic interactions and van
der Waals interactions.
25. A method for detecting ligands comprising: providing a device
capable of detecting ligands, said device comprising at least one
substantially spherical substrate; at least one receptor attached
to said spherical substrate, wherein said at least one receptor is
capable of binding to a ligand to form a receptor-ligand complex
and wherein the formation of said receptor-ligand complex produces
a signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
amplifies said signal upon receptor-ligand complex formation;
exposing a sample containing at least one ligand to said at least
one substrate; allowing said receptor to interact with said at
least one ligand to form at least one receptor-ligand complex; and
measuring the signal produced by said receptor-ligand complex
formation.
26. A device for the detection of ligands comprising: at least one
substantially spherical substrate coated with a receptor-binding
material; at least one receptor attached to said coated spherical
substrate, wherein said at least one receptor is capable of binding
to a ligand to form a receptor-ligand complex and wherein the
formation of said receptor-ligand complex produces a signal; and an
amplification mechanism comprising a liquid crystalline material,
wherein said amplification mechanism amplifies said signal upon
receptor-ligand complex formation.
27. The device of claim 26, wherein said substantially spherical
substrate is non-porous.
28. The device of claim 27, wherein said at least one receptor is
attached to the surface of said non-porous substantially spherical
substrate.
29. The device of claim 26, wherein the substantially spherical
substrate is porous.
30. The device of claim 29, wherein said at least one receptor is
attached to at least one of (i) the surface of said porous
substantially spherical substrate and (ii) the pores of said porous
substantially spherical substrate.
31. The device of claim 30, wherein said at least one receptor is
attached to the surface of said porous substantially spherical
substrate.
32. The device of claim 30, wherein said/at least one receptor is
attached to the pores of said porous substantially spherical
substrate.
33. The device of claim 30, wherein a plurality of receptors are
attached to and randomly distributed on the surface and within the
pores of said porous substantially spherical substrate.
34. The device of claim 26, wherein the liquid crystalline material
is selected from the group consisting of thermotropic liquid
crystalline material and lyotropic liquid crystalline material.
35. The device of claim 34, wherein the liquid crystalline material
is a lyotropic liquid crystalline material.
36. The device of claim 35, wherein the lyotropic liquid
crystalline material is a lyotropic chromonic liquid crystalline
material.
37. The device of claim 34, wherein the liquid crystalline material
is a thermotropic liquid crystalline material.
38. The device of claim 26, wherein the substantially spherical
substrate is made from a material selected from the group
consisting of polymeric and inorganic materials.
39. The device of claim 38, wherein the substantially spherical
substrate is made from a polymeric material.
40. The device of claim 39, wherein the polymeric materials are
selected from the group consisting of polyions, polyalkenes,
polyacrylates, polymethacrylates, polyvinyls, polystyrenes,
polycarbonates, polyesters, polyurethanes, polyamides, polyimides,
polysulfones, polysiloxanes, polysilanes, polyethers, and
polycarboxylates.
41. The device of claim 40, wherein the polymeric material is a
polystyrene.
42. The device of claim 38, wherein the substantially spherical
substrate is made from an inorganic material.
43. The device of claim 42, wherein the inorganic material is
selected from the group consisting of glass, silicon, and colloidal
gold.
44. The device of claim 43, wherein the inorganic material is
glass.
45. The device of claim 26, wherein said at least one receptor is
attached to said spherical substrate by one means selected from the
group consisting of (i) chemical attachment and (ii) physical
attachment.
46. The device of claim 45, wherein said at least one receptor is
attached to said spherical substrate by chemical attachment.
47. The device of claim 46, wherein said chemical attachment is
covalent bonding.
48. The device of claim 45, wherein said at least one receptor is
attached to said spherical substrate by physical attachment.
49. The method of claim 48, wherein said physical attachment is
selected from the group consisting hydrophobic interactions and van
der Waals interactions.
50. A method for detecting ligands comprising: providing a device
capable of detecting ligands, said device comprising at least one
substantially spherical substrate coated with a receptor-binding
material; at least one receptor attached to said spherical
substrate, wherein said at least one receptor is capable of binding
to a ligand to form a receptor-ligand complex and wherein the
formation of said receptor-ligand complex produces a signal; and an
amplification mechanism comprising a liquid crystalline material,
wherein said amplification mechanism amplifies said signal upon
receptor-ligand complex formation; exposing a sample containing at
least one ligand to at least one of said substrate; allowing said
receptor to interact with said at least one ligand to form at least
one receptor-ligand complex; and measuring the signal produced by
said receptor-ligand complex formation.
51. A device for the detection of ligands comprising: a
substantially planar substrate, wherein said substrate is
electrically charged; at least one receptor attached to said
electrically charged substantially planar substrate, wherein said
at least one receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation.
52. The device of claim 51, wherein the liquid crystalline material
is selected from the group consisting of thermotropic liquid
crystalline material and lyotropic liquid crystalline material.
53. The device of claim 52, wherein the liquid crystalline material
is a lyotropic liquid crystalline material.
54. The device of claim 53, wherein the lyotropic liquid
crystalline material is a lyotropic chromonic liquid crystalline
material.
55. The device of claim 52, wherein the liquid crystalline material
is a thermotropic liquid crystalline material.
56. The device of claim 51, wherein the substantially planar
substrate is made from a material selected from the group
consisting of polymeric and inorganic materials.
57. The device of claim 56, wherein the substantially planar
substrate is made from a polymeric material.
58. The device of claim 57, wherein the polymeric materials are
selected from the group consisting of polyions, polyalkenes,
polyacrylates, polymethacrylates, polyvinyls, polystyrenes,
polycarbonates, polyesters, polyurethanes, polyamides, polyimides,
polysulfones, polysiloxanes, polysilanes, polyethers, and
polycarboxylates.
59. The device of claim 58, wherein the polymeric material is a
polystyrene.
60. The device of claim 56, wherein the substantially planar
substrate is made from an inorganic material.
61. The device of claim 60, wherein the inorganic material is
selected from the group consisting of glass, silicon, and colloidal
gold.
62. The device of claim 61, wherein the inorganic material is
glass.
63. The device of claim 51, wherein said at least one receptor is
attached to said substrate by one means selected from the group
consisting of (i) chemical attachment and (ii) physical
attachment.
64. The device of claim 63, wherein said at least one receptor is
attached to said spherical substrate by chemical attachment.
65. The device of claim 64, wherein said chemical attachment is
covalent bonding.
66. The device of claim 63, wherein said at least one receptor is
attached to said spherical substrate by physical attachment.
67. The method of claim 66, wherein said physical attachment is
selected from the group consisting hydrophobic interactions and van
der Waals interactions.
68. A method for detecting ligands comprising: providing a device
capable of detecting ligands, said device comprising at least one
electrically charge substantially planar substrate; at least one
receptor attached to said substrate, wherein said at least one
receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation; exposing a sample containing at least one ligand
to said substrate; allowing said receptor to interact with said at
least one ligand to form at least one receptor-ligand complex; and
measuring the signal produced by said receptor-ligand complex
formation.
69. A device for the detection of ligands comprising: an
substantially planar substrate coated with a receptor-binding
material; at least one receptor attached to said substrate, wherein
said at least one receptor is capable of binding to a ligand to
form a receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation.
70. The device of claim 69, wherein the liquid crystalline material
is selected from the group consisting of thermotropic liquid
crystalline material and lyotropic liquid crystalline material.
71. The device of claim 70, wherein the liquid crystalline material
is a lyotropic liquid crystalline material.
72. The device of claim 71, wherein the lyotropic liquid
crystalline material is a lyotropic chromonic liquid crystalline
material.
73. The device of claim 70, wherein the liquid crystalline material
is a thermotropic liquid crystalline material.
74. The device of claim 69, wherein the substrate is made from a
material selected from the group consisting of polymeric and
inorganic materials.
75. The device of claim 74, wherein the substrate is made from
material a polymeric material.
76. The device of claim 75, wherein the polymeric materials are
selected from the group consisting of polyalkenes, polyacrylates,
polymethacrylates, polyvinyls, polystyrenes, polycarbonates,
polyesters, polyurethanes, polyamides, polyimides, polysulfones,
polysiloxanes, polysilanes, polyethers, and polycarboxylates.
77. The device of claim 76, wherein the polymeric material is
polystyrene.
78. The device of claim 74, wherein the substantially substrate is
made from an inorganic material.
79. The device of claim 78, wherein the inorganic material is
selected from the group consisting of glass, silicon, and colloidal
gold.
80. The device of claim 79, wherein the inorganic material is
glass.
81. The device of claim 69, wherein said at least one receptor is
attached to said substrate by one means selected from the group
consisting of (i) chemical attachment and (ii) physical
attachment.
82. The device of claim 80, wherein said at least one receptor is
attached to said substrate by chemical attachment.
83. The device of claim 81, wherein said chemical attachment is
covalent bonding.
84. The device of claim 80, wherein said at least one receptor is
attached to said substrate by physical attachment.
85. The method of claim 84, wherein said physical attachment is
selected from the group consisting of hydrophobic interactions and
van der Waals interactions.
86. The device of claim 69, wherein the coated substantially planar
substrate is electrically charged.
87. A method for detecting ligands comprising: providing a device
capable of detecting ligands, said device comprising substantially
planar substrate coated with a receptor-binding material; at least
one receptor attached to said substrate, wherein said at least one
receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation; exposing a sample containing at least one ligand
to said substrate; allowing said receptor to interact with said at
least one ligand to form at least one receptor-ligand complex; and
measuring the signal produced by said receptor-ligand complex
formation.
88. The method of claim 87, wherein the coated substantially planar
substrate is electrically charged.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
Ser. No. 09/633,327, filed Aug. 7, 2000, which is a continuation of
U.S. Ser. No. 09/095,196, filed Jun. 10, 1998, now U.S. Pat. No.
6,171,802.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention generally relates to the detection of
a ligand by a receptor. More specifically, the present invention
relates to highly specific receptors and the incorporation of these
receptors into an amplification mechanism comprising a liquid
crystalline material for the rapid and automatic detection of the
ligand, such as microorganisms and products of microorganisms, such
as pathogens and/or their toxins.
BACKGROUND OF THE INVENTION
[0003] The detection of a ligand by a receptor (for example,
detection of a pathogenic agent such as a microbe or toxin by an
antibody; or detection of an antibody in blood by another antibody;
or binding of a chemical toxin, such as nerve gas, to its receptor)
is important in the diagnosis and treatment of individuals exposed
to disease-causing agents. Early detection of pathogenic agents can
be a great benefit in either disease prophylaxis or therapy before
symptoms appear or worsen.
[0004] Every species, strain, or toxin of a microbe contains unique
internal and external ligands. Using molecular engineering and/or
immunological techniques, receptor molecules, such as antibodies,
can be isolated that will bind to these ligands with high
specificity. Methods have also been developed where receptors, such
as antibodies, are linked to a signaling mechanism that is
activated upon binding. Heretofore, however, no system has been
developed that can automatically detect and amplify a receptor
signal coming from the binding of a single or a low number of
ligands in near real time conditions. Such a system is imperative
for rapid and accurate early detection of ligands.
[0005] Many available diagnostic tests are antibody based, and can
be used to detect either a disease-causing agent or a biologic
product produced by the patient in response to the agent. There are
currently three prevailing methods of antibody production for
recognition of ligands (antigens): polyclonal antibody production
in whole animals with recognition for multiple epitopes, monoclonal
antibody production in transformed cell lines with recognition for
a single epitope (after screening), and molecularly engineered
phage displayed antibody production in bacteria with recognition of
a single epitope (after screening). Each of these receptor systems
is capable of binding and identifying a ligand, but the sensitivity
of each is limited by the particular immunoassay detection system
to which it is interfaced.
[0006] Immunoassays, such as enzyme-linked immunosorbent assay
(ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA), are
well known for the detection of antigens. The basic principle in
many of these assays is that an enzyme-, chromogen-, fluorogen-, or
radionucleotide-conjugated antibody permits antigen detection upon
antibody binding. In order for this interaction to be detected as a
color, fluorescence, or radioactivity change, significant numbers
of antibodies must be bound to a correspondingly large number of
antigen epitopes.
[0007] Thus, there is a need for a system that rapidly, reliably,
and automatically detects ligands, especially when present in very
small quantities and consequently provides a measurable signal in
near real time conditions.
SUMMARY OF THE INVENTION
[0008] It is, therefore, an object of the present invention to
provide a device, system, and method that will detect a ligand with
high sensitivity and high specificity in near real time.
[0009] It is another object of the present invention to provide a
device, system, and method that will amplify a signal produced by
the binding of a ligand to a receptor.
[0010] It is a further object of the present invention to provide a
device and system that will distort a surrounding liquid
crystalline material upon the binding of a ligand to a
receptor.
[0011] In general, the present invention provides a system for the
detection and amplification of ligands, such as pathogenic agents,
comprising at least one receptor and an amplification mechanism
comprising a liquid crystalline material coupled to that receptor,
wherein an amplified signal is produced as a result of the receptor
binding the ligand.
[0012] In one embodiment, the present invention provides a device
for the detection of ligands comprising at least one substantially
spherical substrate; at least one receptor attached to said
spherical substrate, wherein said at least one receptor is capable
of binding to a ligand to form a receptor-ligand complex and
wherein the formation of said receptor-ligand complex produces a
signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
amplifies said signal upon receptor-ligand complex formation.
[0013] In another embodiment, the present invention also provides a
method for detecting ligands comprising providing a device capable
of detecting ligands, said device comprising at least one
substantially spherical substrate, at least one receptor attached
to said spherical substrate, wherein said at least one receptor is
capable of binding to a ligand to form a receptor-ligand complex
and wherein the formation of said receptor-ligand complex produces
a signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
comprises a liquid crystalline material and amplifies said signal
upon receptor-ligand complex formation; exposing a sample
containing at least one ligand to at least one substrate; allowing
said receptor to interact with said at least one ligand to form at
least one receptor-ligand complex, and measuring the signal
generated by said receptor-ligand complex formation.
[0014] In another embodiment, the present invention further
provides a device for the detection of ligands comprising: at least
one substantially spherical substrate coated with a
receptor-binding material; at least one receptor attached to said
coated spherical substrate, wherein said at least one receptor is
capable of binding to a ligand to form a receptor-ligand complex
and wherein the formation of said receptor-ligand complex produces
a signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
amplifies said signal upon receptor-ligand complex formation.
[0015] The present invention also provides a method for detecting
ligands comprising: providing a device capable of detecting
ligands, said device comprising at least one substantially
spherical substrate coated with a receptor-binding material; at
least one receptor attached to said coated spherical substrate,
wherein said at least one receptor is capable of binding to a
ligand to form a receptor-ligand complex and wherein the formation
of said receptor-ligand complex produces a signal; and an
amplification mechanism comprising a liquid crystalline material,
wherein said amplification mechanism amplifies said signal upon
receptor-ligand complex formation; exposing a sample containing at
least one ligand to at least one of said substrate; allowing said
receptor to interact with said at least one ligand to form at least
one receptor-ligand complex; and measuring the signal produced by
said receptor-ligand complex formation.
[0016] The present invention further provides a device for the
detection of ligands comprising: a substantially planar substrate,
wherein said substrate is electrically charged; at least one
receptor attached to said charged substrate, wherein said at least
one receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation.
[0017] The present invention further includes a method for
detecting ligands comprising: providing a device capable of
detecting ligands, said device comprising at least one electrically
charged substantially planar substrate; at least one receptor
attached to said substrate, wherein said at least one receptor is
capable of binding to a ligand to form a receptor-ligand complex
and wherein the formation of said receptor-ligand complex produces
a signal; and an amplification mechanism comprising a liquid
crystalline material, wherein said amplification mechanism
amplifies said signal upon receptor-ligand complex formation;
exposing a sample containing at least one ligand to said substrate;
allowing said receptor to interact with said at least one ligand to
form at least one receptor-ligand complex; and measuring the signal
produced by said receptor-ligand complex formation.
[0018] The present invention further provides a device for the
detection of ligands comprising: a substantially planar substrate
coated with a receptor-binding material; at least one receptor
attached to said coated substrate, wherein said at least one
receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation.
[0019] The present invention also provides a method for detecting
ligands comprising: providing a device capable of detecting
ligands, said device comprising substantially planar substrate
coated with a receptor-binding material; at least one receptor
attached to said coated substrate, wherein said at least one
receptor is capable of binding to a ligand to form a
receptor-ligand complex and wherein the formation of said
receptor-ligand complex produces a signal; and an amplification
mechanism comprising a liquid crystalline material, wherein said
amplification mechanism amplifies said signal upon receptor-ligand
complex formation; exposing a sample containing at least one ligand
to said substrate; allowing said receptor to interact with said at
least one ligand to form at least one receptor-ligand complex; and
measuring the signal produced by said receptor-ligand complex
formation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A is a schematic representation of the lamellar
structure of a lyotropic liquid crystal formed by alternating
layers of water and biphilic molecules.
[0021] FIG. 1B is a schematic representation of the amplification
mechanism with a receptor inserted into the lyotropic liquid
crystal.
[0022] FIG. 1C is a schematic representation of the amplification
mechanism with the specific ligand bound to its receptor causing
deformation of the liquid crystal and alteration of the
transmission of polarized light.
[0023] FIG. 2A is a representation of a non-porous (solid)
spherical substrate having a plurality of receptors attached to the
outer surface of the sphere.
[0024] FIG. 2B is a representation of a porous spherical substrate
having a plurality of receptors attached to the outer surface of
the sphere and within the pores of the sphere.
[0025] FIG. 2C is a representation of a non-porous (solid)
spherical substrate having a plurality of receptors attached to the
outer surface of the sphere with ligand bound to a portion of the
receptors.
[0026] FIG. 2D is a representation of a porous spherical substrate
having a plurality of receptors attached to the outer surface of
the sphere and within the pores of the sphere with ligand bound to
a portion of the receptors.
[0027] FIG. 3A is a representation of, a non-porous (solid)
spherical substrate having a plurality of receptors attached to the
outer surface of the sphere showing the liquid crystalline material
orientation about the receptor-bound sphere.
[0028] FIG. 3B is a representation of, a porous spherical substrate
having a plurality of receptors attached to the outer surface of
the sphere and within the pores of the sphere showing the liquid
crystalline material orientation about the receptor-bound
sphere.
[0029] FIG. 3C is a representation of a non-porous (solid)
spherical substrate having a plurality of receptors attached to the
outer surface of the sphere with ligand bound to a portion of the
receptors showing the change in liquid crystalline material
orientation about the sphere when ligand is bound.
[0030] FIG. 3D is a representation of a porous spherical substrate
having a plurality of receptors attached to the outer surface of
the sphere and within the pores of the sphere with ligand bound to
a portion of the receptors showing the change in liquid crystalline
material orientation about the sphere when ligand is bound.
[0031] FIG. 4A is a graph showing the number of light transmissive
microdomains in the neutral grey liquid crystalline material using
(a) polycarboxylate microspheres coated with anti-E.coli antibody
and (b) polycarboxylate microspheres coated with Bovine Serum
Albumin (BSA). The open circles (o) represent the number of light
transmissive microdomains in the neutral grey liquid crystalline
material using polycarboxylate microspheres coated with anti-E.coli
antibody, and the filled in circles (.circle-solid.)represents the
number of light transmissive microdomains in the neutral grey
liquid crystalline material using polycarboxylate microspheres
coated with BSA.
[0032] FIG. 4B is a graph showing the number of light transmissive
microdomains in the neutral grey liquid crystalline material using
(a) polystyrene microspheres coated with anti-E.coli antibody and
(b) polystyrene microspheres coated with Bovine Serum Albumin. The
open circles (o) represent the number of light transmissive
microdomains in the neutral grey liquid crystalline material using
polystyrene microspheres coated with anti-E.coli antibody, and the
filled in circles (.circle-solid.)represents the number of light
transmissive microdomains in the neutral grey liquid crystalline
material using polystyrene microspheres coated with (BSA).
[0033] FIG. 5A is a graph showing the number of light transmissive
microdomains in the disodium cromoglycate liquid crystalline
material using (a) polycarboxylate microspheres coated with
anti-E.coli antibody and (b) polycarboxylate microspheres coated
with Bovine Serum Albumin. The open circles (o) represent the
number of light transmissive microdomains in the disodium
cromoglycate liquid crystalline material using polycarboxylate
microspheres coated with anti-E.coli antibody, and the filled in
circles (.circle-solid.)represents the number of light transmissive
microdomains in the disodium cromoglycate liquid crystalline
material using polycarboxylate microspheres coated with BSA.
[0034] FIG. 5B is a graph showing the number of light transmissive
microdomains in the disodium cromoglycate liquid crystalline
material using (a) polystyrene microspheres coated with anti-E.coli
antibody and (b) polystyrene microspheres coated with Bovine Serum
Albumin. The open circles (o) represent the number of light
transmissive microdomains in the disodium cromoglycate liquid
crystalline material using polystyrene microspheres coated with
anti-E.coli antibody, and the filled in circles
(.circle-solid.)represents the number of light transmissive
microdomains in the disodium cromoglycate liquid crystalline
material using polystyrene microspheres coated with BSA.
[0035] FIG. 6A is a representation of a planar substrate having a
plurality of receptors attached to one surface of the substrate and
without ligand bound to the receptors.
[0036] FIG. 6B is a representation of a substantially planar
substrate having a plurality of receptors attached to one surface
of the substrate and with some ligands bound to a portion of the
receptors.
[0037] FIG. 7A is a representation of a planar substrate having a
plurality of receptors attached to one surface of the substrate
without ligand bound to the receptors showing the liquid
crystalline material orientation when ligand is not bound to
receptor.
[0038] FIG. 7B is a representation of a planar substrate having a
plurality of receptors attached to one surface of the substrate
with some ligands bound to a portion of the receptors showing the
change in liquid crystalline material orientation when ligand is
bound to receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0039] In the present invention, ligand-specific receptors are
interfaced with an amplification mechanism such that a
receptor-ligand interaction forms birefringent receptor-ligand
aggregates and/or changes the conformation of the receptor and
produce a light transmissive signal. Amplification preferably
occurs through a birefringent shift that can be photometrically
detected. The detected signal may then be electronically amplified
to automate the system.
Ligand Detection Component
[0040] Any receptor, such as antibodies or biologic/biologically
engineered receptors for ligands, can be incorporated into the
device as long as binding of the ligand to the receptor causes a
detectable ligand aggregation and/or distortion (change in
conformation) of the receptor. For example, any type of
monospecific antibody (polyclonal, monoclonal, or phage displayed)
can effectively function as a receptor and, thus, each of those
antibody types will be described in the following paragraphs.
Although phage-displayed antibodies can be expeditiously modified
for identification of new ligands and are used as receptor examples
in this patent application, any physically-distortable
receptor-ligand interaction is appropriate for the detection
component.
[0041] Polyclonal antibodies: Antibody-based antigen detection has
been exploited for several decades. Injection of a purified ligand
(antigen) into a host animal stimulates the immune system to
produce an array of antibodies against various reactive sites on
the antigen. Since several lymphocytes are responding to different
antigenic epitopes, a multi-specific antibody cocktail (polyclonal)
is created and can be purified for antigen detection.
[0042] Monoclonal antibodies: Antibody-producing spleen cells (B
lymphocytes) are fused with immortalized myeloma cells to create
hybridomas which provide nearly infinite quantities of antibody
with a single, defined specificity. Interstrain and even
interspecies hybrids of these `monoclonal` antibodies can be
generated through genetic engineering techniques. These highly
specific antibodies have significant therapeutic potential, as
evidenced by the U.S. Food and Drug Administration's approval of
the use of mouse-human chimeric antibodies for treatment of
selected diseases.
[0043] Phage-displayed mono-specific antibodies: Phage-displayed
techniques will be used to isolate single chain chimeric antibodies
to various pathogenic agents. The genomic DNA of the B lymphocyte
contains the code to produce an antibody to virtually all possible
ligands (antigens). In a phage displayed antibody system (PDA), DNA
encoding a single chain chimnera of the native antibody's
hypervariable ligand-binding region is synthesized by joining DNA
encoding an antibody heavy chain and DNA encoding an antibody light
chain and inserting therebetween DNA encoding a linker region. The
desired amino acid sequence of the linker region depends on the
characteristics required for any given amplification mechanism. The
linker region may have to be able to interact and/or bond to a
protein or other substance. Therefore, the polypeptide sequence may
have to have, for example, a particular conformation, specifically
placed functional groups to induce ionic or hydrogen bonds, or a
hydrophobicity that is compatible with the amplification mechanism.
Regardless of the type of amplification mechanism, however, the
linker region plays a critical role in interfacing the
amplification mechanism to the receptor.
[0044] The DNA, preferably human or mouse, encoding the single
chain chimeric antibody is cloned into a bacteriophage (phage)
vector using well-known techniques (Marks et al., J. Mol. Bio. Vol.
222:581 (1991); Griffiths et al., EMBO J. 12:725 (1993); and
Winters et al., Ann. Rev. Immunol. 12:433 (1994)), incorporated
herein by reference. The single chain chimeric antibodies then
become displayed on the surface of a filamentous phage with the
hypervariable antigen-binding site extended outward.
[0045] After the addition of ligands, phage that are reactive
against non-targeted ligands are subtracted from the phage library
using known techniques (Marks et al., J. Mol. Bio. Vol. 222:581
(1991); Griffiths et al., EMBO J. 12:725 (1993); and Winters et
al., Ann. Rev. Immunol. 12:433 (1994)), incorporated herein by
reference. The remaining phage are reacted with their specific
ligand and phage reactive with that specific ligand eluted. Each of
these phage are then isolated and expressed in a bacterial host,
such as Escherichia coli (E. coli) to produce a large quantity of
phage containing the desired surface-displayed antibody. Each of
the aforementioned methods relating to synthesizing and cloning
DNA, subtracting phages, isolating and expressing phages and
recovering viral DNA are well known and fully described by Marks et
al., J. Mol. Biol. (1991); Griffiths et al., EMBO J. 12:725 (1993);
and Winters et al., Ann. Rev. Immunol. 12:433 (1994), all of which
are incorporated herein by reference.
Amplification Component
[0046] An amplification mechanism including liquid crystalline
material is utilized to amplify a ligand-receptor complex, thereby
detecting the presence of ligands in a sample.
[0047] A liquid crystal is a state of matter in which molecules
exhibit some orientational order but little positional order. This
intermediate ordering places liquid crystals between solids (which
possess both positional and orientational order) and isotropic
fluids (which exhibit no long-range order). Solid crystal or
isotropic fluid can be caused to transition into a liquid crystal
by changing temperature (creating a thermotropic liquid crystal) or
by using an appropriate diluting solvent to change the
concentration of solid crystal (creating a lyotropic liquid
crystal). Both thermotropic and lyotropic liquid crystals can be
used as the amplification mechanism of the device of the present
invention. In one embodiment, chromonic lyotropic liquid
crystalline material are used as the amplification component of the
device of the present invention.
[0048] Among these non-surfactant lyotropic liquid crystals are
so-called lyotropic chromonic liquid crystals (LCLCs). The LCLC
family embraces a range of dyes, drugs, nucleic acids, antibiotics,
carcinogens, and anti-cancer agents. For a review of lyotropic
chromonic liquid crystals see J. Lydon, Chromonics, in: Handbook of
Liquid Crystals, Wiley-VCH, Weinheim, vol. 2B, p. 981 (1998). The
LCLCs are fundamentally different from the better known
surfactant-based lyotropic systems. Without limitation, one
difference is that LCLC molecules are disc-like or plank-like
rather than rod-like. The polar hydrophilic parts form the
periphery, while the central core is relatively hydrophobic. This
distinction creates a range of different ordered structures.
Individual disc-like molecules may form cylindrical aggregates in
water. The LCLCs are assumed to be formed by elongated aggregates,
lamellar structures, and possibly by aggregates of other
shapes.
[0049] As seen in FIG. 1A, most lyotropic liquid crystals,
designated generally by the numeral 1, are formed using water 2 as
a solvent for biphilic molecules 3, for example, molecules which
possess polar (hydrophilic) parts 4 and a polar (hydrophobic) parts
5. When water 2 is added to biphilic molecules 3, a bilayer 6 forms
as the hydrophobic regions coalesce to minimize interaction with
water 2 while enhancing the polar component's interaction with
water. The concentration and geometry of the specific molecules
define the supramolecular order of the liquid crystal. The
molecules can aggregate into lamellae as well as disk-like or
rod-like micelles, or, generally, aggregates of anisometric shape.
These anisometric aggregates form a nematic, smectic, columnar
phase, of either non-chiral or chiral (cholesteric phase) nature.
For example, the molecules form a stack of lamellae of alternating
layers of water and biphilic molecules, thus giving rise to a
lamellar smectic phase.
[0050] Lyotropic liquid crystals are usually visualized as ordered
phases formed by rod-like molecules in water. A fundamental feature
of the surfactant molecules is that the polar hydrophilic head
group has an attached flexible hydrophobic tail. There is, however,
a variety of other lyotropic systems that are not of the surfactant
type, but which can also be successfully used in the present
invention.
[0051] Liquid crystalline phases are characterized by orientational
order of molecules or their aggregates. In the uniaxial liquid
crystal phases such as nematic and smectic A, the average direction
of orientation of the molecules or aggregates is described by a
unit vector, called the director and denoted n. Generally, the two
opposite directions of the director are equivalent, n=-n. In the
uniaxial phases, the director is simultaneously the optical axis of
the medium. An optically uniaxial liquid crystalline medium is
birefringent. A uniaxial birefringent medium is characterized by
two optical refractive indices: an ordinary refractive index
"n.sub.o" for an ordinary wave and an extraordinary refractive
index "n.sub.e" for an extraordinary wave.
[0052] When the liquid crystal is viewed between two crossed
polarizers, the appearing texture and the intensity of transmitted
light are determined by orientation of the optical axis (director)
with respect to the polarizers and other factors, as clarified
below.
[0053] Consider, as an example, a nematic slab sandwiched between
two glass plates and placed between two crossed polarizers. We
follow the description given by M. Kleman and O. D. Lavrentovich,
"Soft Matter Physics: An Introduction," Springer-Verlag New York,
(2001). The director n is in plane of the slab and depends on the
in-plane coordinates (x,y). We assume that it does not depend on
the vertical coordinate z. The light beam impinges normally on the
cell, along the axis z. A polarizer placed between the source of
light and the sample makes the impinging light linearly polarized.
In the nematic, the linearly polarized wave of amplitude A
intensity I.sub.0=A.sup.2 and the frequency .omega. splits into the
ordinary and extraordinary waves with mutually perpendicular
polarizations and amplitudes Asin.beta. and Acos.beta.,
respectively; .beta.(xy) is the angle between the local n(x,y) and
the polarization of incident light. The vibrations of the electric
vectors at the point of entry are in phase. However, the two waves
take different times, n.sub.od/c and n.sub.ed/c, respectively, to
pass through the slab. Here d is the thickness of the slab, and c
is the speed of light in vacuum. At the exit point, the electric
vibrations 1 ~ A sin cos ( t - 2 0 n o d ) and ~ A cos cos ( t - 2
0 n e d )
[0054] gain a phase shift 2 = 2 d 0 ( n e - n o ) ,
[0055] where .lambda..sub.0 is the wavelength in vacuum. The
projections of these two vibrations onto the polarization direction
of the analyzer behind the sample are 3 a = A sin cos cos ( t - 2 0
n o d ) and b = - A sin cos cos ( t - 2 0 n e d ) (Eq.1)
[0056] When two harmonic vibrations
A.sub.1cos((.omega.t+.PHI..sub.1) and
A.sub.2cos(.omega.t+.PHI..sub.2) of the same frequency occur along
the same directions, then the resulting vibration {overscore
(A)}cos(.omega.t+{overscore (.PHI.)}) has an amplitude defined from
{overscore
(A)}.sup.2=A.sub.1.sup.2+A.sub.2.sup.2+2A.sub.1A.sub.2cos(.PHI-
..sub.1-.PHI..sub.2). The analyzer thus transforms the pattern of
(x,y)-dependent phase difference into the pattern of transmitted
light intensity I(x,y)={overscore (A)}.sup.2. The intensity of
light passed through the crossed polarizers and the nematic slab
between them follows from Eq.(1) as 4 I = I 0 sin 2 2 sin 2 [ d 0 (
n e - n o ) ] . (Eq.2)
[0057] The last formula refers to the case when n is perpendicular
to the axis z. If n makes an angle .theta. with the axis z, then
(2) becomes 5 I = I 0 sin 2 2 sin 2 [ d 0 ( n o n e n e 2 cos 2 + n
o 2 sin 2 - n o ) ] (Eq.3)
[0058] Below is a representation of the propagation of light
through a polarizer, uniaxial slab and analyzer. 1
[0059] The treatment can be further extended to describe the
optical properties of complex director configurations, for example,
in the electric field-driven cells. However, for the case when the
director is distorted by ligand-receptor interactions rather than
by an externally applied electric or magnetic field equations (2)
and (3) are fundamental for understanding liquid crystal textures.
First, note that the phase shift and thus I depend on
.lambda..sub.0. As a result, when the sample is illuminated with a
white light, it would show a colorful texture. The interference
colors are especially pronounced when
(n.sub.e-n.sub.o)d.about.(1.div.3).lambda..sub.0. With typical
(n.sub.e-n.sub.o).about.0.2, .lambda..sub.0.about.500 nm, the
`colorful` range of thicknesses is d.about.(1.div.10).mu.m. Second,
the director tilt .theta. greatly changes the phase shift. When
n.parallel.z (the so-called homeotropic orientation, .theta.=0, the
sample looks dark: only the ordinary wave propagates and, according
to Eq. (3), I=0. Third, if .theta.=0 but .beta.=0, .pi./2, . . . ,
one might still observe dark textures, I=0, even in
non-monochromatic light. In a sample with in-plane director
distortions n(x,y), wherever n (or its horizontal projection) is
parallel or perpendicular to the polarizer, the propagating mode is
either pure extraordinary or pure ordinary and the corresponding
region of the texture appears dark. By aligning a well-oriented
liquid crystal sample between two crossed polarizers, one can find
an "extinction" position in which the sample is dark. This
extinction position corresponds to the director aligned along the
polarization direction of polarizer or analyzer, .beta.=0, .pi./2,
. . . , .
[0060] The extinction state will occur for all points of the
sample, as long as the director field is not perturbed and uniform.
However, if the director field is disturbed and varies from point
to point within the slab, then the condition of extinction (meaning
I=0 in Equations (2) and (3)) cannot be satisfied everywhere and
the resulting intensity of light passing through the polarizer,
liquid crystal slab and analyser will be different from zero. Such
a disturbance of the liquid crystal detector can be caused by the
receptor-ligand interaction, if this interaction realigns the
liquid crystalline molecules or aggregates in the neighborhood.
These are the important features allowing us to use the liquid
crystals as detection and amplification system.
[0061] Most biologic receptors possess both hydrophilic and
hydrophobic regions and, thus, readily incorporate into biphilic
lyotropic liquid crystals. Additionally, the inactivated receptors
do not destroy the optical anisotropy (birefringence) of the liquid
crystal and, therefore, the device comprised of a receptor-enriched
liquid crystal with a following analyzer remains nontransparent to
polarized light when proper alignment satisfies the condition of
extinction, as seen from equations (2) and (3). In this case, light
would be able to pass through the liquid crystal but the analyzer
would not let light pass any further, because the polarization of
the light will be perpendicular to the plane of polarization of the
analyzer. However, director orientation and, thus, the orientation
of optical axis is disrupted when receptor conformation shifts as
during the formation of the receptor-ligand complex. The elasticity
of the liquid crystal enhances the local distortions in the
vicinity of the receptor-ligand complex, and expands it to an
optically detectable, supramicron scale. These distortions
generally deviate the director from the "extinction" orientations
such as .beta.=0,.+-..pi./2, . . . , and make the system locally
transparent, as the light beam is not blocked by the analyzer.
Configurations of Ligand Detection Device
[0062] By way of example, one envisioned application of the present
invention is in a multiwell system. Each well of the system would
contain PDAs to a specific ligand, such as a pathogenic microbe,
interfaced with an amplification mechanism of the present
invention. When the microbial agent interacts with the antibody,
the resulting antibody distortion triggers the amplification
mechanism. Preferably, the amplified signal is then transduced into
a perceptible signal. Accordingly, it is envisioned that such a
system could be placed in a physician's -office, and be used in
routine diagnostic procedures. Alternatively, such a system could
be placed on or near soldiers in battle, and the invention used to
alert the soldiers to the presence of a toxic agent. It is further
envisioned that a multiwell system, is preferably used in
conjunction with the liquid crystal embodiment described
herein.
[0063] Thus, in one embodiment of the present invention, shown
schematically in FIGS. 1B and 1C, a lyotropic liquid crystalline
material is used as an amplification mechanism. As shown in FIG.
1B, the device consists of a light source 10, an initial polarizer
12, with the direction of polarization in the plane of the figure,
a pathogen detection system 14a, comprising monospecific antibodies
14b embedded in biphilic, lyotropic liquid crystalline material
14c, a secondary polarizer 16, with the direction of polarization
perpendicular to the plane of the figure, and a photodetector
18.
[0064] In operation, the initial polarizer 12 organizes a light
beam 22 that is linearly polarized in the plane of the figure. The
optical axis 20 of the inactivated device is perpendicular to the
pathogen detection system 14a, and thus no birefringence of the
transluminating linearly polarized light stream 22 occurs. Since
the polarization direction of the secondary polarizer 16 is
perpendicular to the transluminating linearly polarized light 22,
the secondary polarizer prevents light from reaching the
photodetector 18.
[0065] Binding of a ligand 24, such as a microbe, to the receptor
14b, such as an antibody, distorts the liquid crystal 14c, and thus
causes detectable changes in the light transmitted through the
sample between two crossed polarizers. This activation process is
illustrated in FIG. 1C. The receptor (antibody) 14b is embedded in
the lyotropic liquid crystal 14c. The spacial distortion caused by
the formation of the antigen-antibody complex is transmitted to the
contiguous liquid crystal 14c. The elastic characteristics of the
liquid crystal permit the distortion to be transmitted over a
region much larger than the size of the receptor-ligand complex.
This allows the use of the standard optical phenomenon of
birefringence to detect distortions caused by the receptor-ligand
complex, see Max Born and E. Wolf., Principals of Optics, Sixth
edition, Pressman Press, Oxford, 1980), as well as the discussion
above. The altered liquid crystalline order distorts the optical
axis and induces changes in the transmitted light, as discussed
above. For example, if the sample is originally aligned in the
`extinction" position (so that .beta.0 or .beta.=.pi./2), the
transmission of light through the two crossed polarizers and a
sample between them is zero. The distortions caused by the
receptor-ligand complex violate the condition of complete
extinction since these distortions deviate the angle .beta. from
the values .beta.=0 and/or .beta.=.pi./2. Therefore, the
transmittance of the light through the pair of polarizers and the
liquid crystal sample will be different from zero in the regions of
sample where the distortions occur. The secondary polarizer
(analyzer) 16 allows this portion of light to pass to the
photodetector 18. The detected change or amplification in light
intensity can be transduced electronically into a perceptible
signal.
[0066] In one preferred embodiment, the device of the present
invention may include at least one substantially spherical
substrate to which receptors may be attached. The receptor or
receptors that are attached to the spherical substrate must be
capable of binding to a desired ligand to form a receptor-ligand
complex such that, upon formation of said receptor-ligand complex a
signal is produced. An amplification mechanism is interfaced with
the receptor-ligand complex, where the amplification mechanism
amplifies the signal produced by receptor-ligand complex
formation.
[0067] The substantially spherical substrate utilized in the
present invention can be non-porous (solid) or porous. In one
embodiment, the substantially spherical substrate is a solid sphere
and the at least one receptor is attached to the outer surface of
the spherical substrate.
[0068] In another embodiment, the substantially spherical substrate
is porous. According to this embodiment, the at least one receptor
may be attached to either the surface of said porous substantially
spherical substrate, the pores of said porous substantially
spherical substrate, or both. By way of non-limiting example, if
only one receptor is attached to the substantially spherical
substrate, then the receptor can be attached to either the outer
surface of the porous sphere or in the pores of the sphere. In
embodiment having more than one receptor attached to the spherical
substrate, then the receptors can all be attached to the outer
surface of the sphere, all the receptor can be attached within the
pores of the sphere, or some receptors can be attached to the outer
surface of the sphere and other receptors can be attached to the
pores of the sphere. The use of a porous sphere or bead provides a
greater surface area on which to attach receptors and, therefore,
would also permit surface and luminal receptor-ligand
interactions.
[0069] The receptors may be attached to the spherical substrate in
any manner known in the art, including chemical attachment and
physical attachment. In one preferred embodiment, the receptors are
attached to the spherical substrate by a chemical attachment, such
as by covalent bonding to sulfate, amine, carboxyl or hydroxyl
groups imbedded in the spherical substrate. However, it should be
noted that the receptors wherein said at least one receptor is
attached to said spherical substrate by any means of physical
attachment.
[0070] The substantially receptor-coated spherical substrate is
made from a material including, but not limited to, polymeric and
inorganic materials. In one preferred embodiment, the substantially
receptor-coated spherical substrate is comprised of a polymeric
material. Suitable polymeric materials which may comprise the
spherical substrate include, but are not limited to, polyalkenes,
polyacrylates, polymethacrylates, polyvinyls, polystyrenes,
polycarbonates, polyesters, polyurethanes, polyamides, polyimides,
polysulfones, polysiloxanes, polysilanes, polyethers, polycations,
polyanions, and polycarboxylates. One particularly useful polymeric
material used to manufacture the spherical substrate is
polystyrene, especially when modified with copolymers of acrylic
ester, chloromethylstyrene, methylolamine, methyl methacrylate or
made zwitterionic. If a polycation is utilized as the material of
the spherical substrate, one particularly suitable polycation is
poly(diallyldimethylammoniumchloride).
[0071] In another embodiment, the substantially receptor-coated
spherical substrate is made from an inorganic material. Suitable
inorganic materials include, but are not limited to, glass,
silicon, and colloidal gold. In one preferred embodiment, the
spherical substrate is a glass bead.
[0072] The liquid crystalline material that is utilized with the
substantially coated spherical substrate includes all known types
of thermotropic liquid crystalline materials and lyotropic liquid
crystalline materials. In one preferred embodiment, lyotropic
liquid crystalline material is used as the amplification mechanism.
In another embodiment, lyotropic liquid crystalline materials of
different origin, including surfactant and lyotropic chromonic
liquid crystalline material, may used with the spherical
substrate.
[0073] As described herein above, any receptor, such as antibodies
or biologic/biologically engineered receptors for ligands, can be
incorporated into the device as long as binding of the ligand to
the receptor produces a detectable signal. Therefore, any type of
monospecific antibody, including all polyclonal, monoclonal, or
phage displayed antibodies can effectively function as a
receptor.
[0074] In another embodiment, the present invention provides a
method for detecting ligands. The method for detecting ligands,
according this embodiment, includes providing a device that
comprises at least one substantially spherical substrate, at least
one receptor attached to said spherical substrate, and an
amplification mechanism. The at least one receptor must be capable
of binding to a ligand to form a receptor-ligand complex and, upon
formation of the receptor-ligand complex, a signal is produced. The
amplification mechanism must be capable of amplifying the signal
produced by the receptor-ligand complex formation. Generally, a
sample containing ligands specific to the receptor that is attached
to the sphere is exposed to the device. After exposing the
ligand-containing sample to the device, the receptor or plurality
of receptors that are attached to the sphere are allowed to
interact with the ligands in the sample to form at least one
receptor-ligand complex. The formation of the receptor-ligand
complex produces a detectable signal. The signal generated by the
formation of the receptor-ligand complex is amplified by the
amplification mechanism, namely, the liquid crystalline material.
The amplified signal may then be measured and quantitated by those
known methods easily determined by those having ordinary skill in
the art.
[0075] In one embodiment, the measurement and quantitation of the
of the receptor-ligand complex formation is mediated in the fluid
phase or "flow through" phase, whereby the spheres and the liquid
crystalline material are injected through an optical device that
can determine the orientation of the liquid crystalline material.
Utilizing this particular method of quantitation permits "field
capture" of ligands using previously prepared spherical beads
having a predetermined receptor attached thereto. Thus, for
example, the ligands can be captured "in the field", transported,
and analyzed at the later time. This method obviates the need for
special transport media usually required to "protect" the ligand
until detection is performed.
[0076] In another embodiment, the device for the detection of
ligands comprises at least one substantially spherical substrate
coated with a receptor-binding or receptor-crosslinking material,
at least one receptor attached to the coated spherical substrate,
and an amplification mechanism comprising a liquid crystalline
material. The at least one receptor is capable of binding to a
ligand to form a receptor-ligand complex and the formation of the
receptor-ligand complex produces a signal. The signal produced is
then amplified by the amplification mechanism upon receptor-ligand
complex formation. According to the present embodiment, the
crosslinker material may be, without limitation, natural or
synthetic polymers, proteins, and secondary antibodies.
[0077] In one preferred embodiment, molecules with specificity for
receptors, such as the specificity exhibited by Protein A, Protein
G or anti-immunoglobulin antibodies for immunoglobulins, will be
chemically cross linked to the spherical substrate. Receptors with
specificity for unique pathogens, toxins or proteins will then be
bound to the immobilized molecules.
[0078] In another embodiment, the present invention provides a
method for detecting ligands comprising providing a device capable
of detecting ligands. According to this embodiment, the device
comprises at least one substantially spherical substrate coated
with a receptor-binding material; at least one receptor attached to
said spherical substrate, and an amplification mechanism comprising
a liquid crystalline material. The at least one receptor is capable
of binding to a ligand to form a receptor-ligand complex and upon
the formation of a receptor-ligand complex produces a signal. The
amplification mechanism amplifies said signal upon receptor-ligand
complex formation. The method includes exposing a sample containing
at least one ligand to at least one of said substrate and allowing
the receptor to interact with the ligands in the sample to form at
least one receptor-ligand complex. The signal produced by said
receptor-ligand complex formation is then measured.
[0079] In another preferred embodiment, the device for detecting
ligands comprises an electrically charged, substantially planar
substrate, at least one receptor attached or bound to the planar
substrate, and an amplification mechanism including a liquid
crystalline material.
[0080] As described above the spherical substrates, the liquid
crystalline material that is utilized with the substantially coated
spherical substrate includes all known types of thermotropic liquid
crystalline materials and lyotropic liquid crystalline materials.
In a preferred embodiment, lyotropic liquid crystalline materials
are used with the electrically charged substrate. In another
preferred embodiment, lyotropic chromonic liquid crystalline
material is utilized.
[0081] In another embodiment, a method for detecting ligands is
disclosed comprising providing a device capable of detecting
ligands, the device comprising at least one electrically charge
substantially planar substrate, at least one receptor attached to
the substrate, and an amplification mechanism comprising a liquid
crystalline material. The at least one receptor is capable of
binding to a ligand to form a receptor-ligand complex and the
formation of a receptor-ligand complex produces a signal. A sample
containing ligands is exposed to the receptor coated substrate, and
is allowed to interact with the receptors to form at least one
receptor-ligand complex. The signal produced by the receptor-ligand
complex formation. is amplified by the liquid crystalline
amplification mechanism.
[0082] The present invention also provides a device for the
detection of ligands including an electrically charged,
substantially planar substrate, at least one receptor and an
amplification mechanism. The at least one receptor attached to the
charged substrate is capable of binding to a ligand to form a
receptor-ligand complex. The formation of the receptor-ligand
complex produces a detectable signal, which is amplified by the
amplification mechanism comprising a liquid crystalline
material.
[0083] A charged substrate may be formed by depositing a polyionic
material from an aqueous solution onto the substrate. Without
limitation, for example, poly(diallyldimethylammoniumchloride)
becomes positively charged in aqueous solutions as negatively
charged Cl atoms dissociate from the molecule. To deposit the
polyion layer onto a glass substrate, the substrate should be
cleaned and then dipped it into the aqueous solution of the
polyion. The polyion adsorbs to the surface of the substrate. The
excess of the polyion can be washed out with an aqueous solution.
In one preferred embodiment, an electrically charged spherical
substrate is utilized with lyotropic chromonic liquid crystals.
According to this embodiment, the opposite electric charges of the
polyionic substrate and the chromonic liquid crystalline molecules
are kept in close contact by electrostatic forces.
[0084] In another embodiment, the present invention further
provides a device for the detection of ligands comprising an
substantially planar substrate coated with a receptor-binding or
crosslinking material, at least one receptor, and an amplification
mechanism. The at least one receptor attached to the coated
substrate is capable of binding to a ligand to form a
receptor-ligand complex. The formation of the receptor-ligand
complex produces a signal, which is amplified by the amplification
mechanism comprising a liquid crystalline material. As described
above for spherical substrates, the planar substrate is coated with
molecules having specificity for receptors that include, without
limitation, polymers, Protein A, Protein G, anti-immunoglobulin
antibodies for immunoglobulins. Receptors with specificity for
unique pathogens, toxins, or proteins will then be bound to the
immobilized receptor-binding or crosslinker molecules coated on the
surface of the substrate.
[0085] In a variation of the is embodiment, the coated
substantially planar substrate may also be electrically charged by
any suitable means.
[0086] In one preferred embodiment, when utilizing any of the above
described ligand detection and amplification devices, the
non-specific aggregates are removed from the ligand containing
sample prior to reacting the ligands with receptor and measuring
the signal produced. The non-specific aggregates may be removed by
any suitable means including, but not limited to, filtering. The
filtered sample will then be reacted with the desired receptor and
the resulting signal produced by the formation of receptor-ligand
complex will be amplified by the liquid crystalline material and
measured. Without being bound to any particular theory, it is
thought that the presence of the large non-specific aggregates will
increase light transmission through the liquid crystalline material
and may, therefore, produce false positive signals.
EXAMPLES
[0087] The following examples demonstrate the use of one embodiment
of the present invention, namely, substantially receptor-coated
microspheres with the liquid crystal amplification mechanism to
detect and amplify ligands upon receptor-ligand complex formation.
A ligand detection system was created by introducing into the
liquid crystal amplification mechanism a desired quantity of
microspheres whose surface was substantially coated with
microbe-specific antibodies. The examples are intended for
illustrative purposes only, and should not be construed as limiting
the scope of the present invention in any manner.
[0088] The devices were evaluated by inserting antibody-coated
microspheres into a lyotropic liquid crystal. For each assay, 10
.mu.l of serially diluted microspheres (coated with either the
anti-E. coli K99 antibody or BSA) was mixed with 10 .mu.l of the
stock E. coli solution and incubated for 30 minutes at room
temperature. The 20% stock solution of liquid crystal (50 .mu.l)
was added to the microsphere-antibody solution and gently mixed
prevent the formation of bubbles. A 60 .mu.l fraction of the
mixture was deposited on a clean, polymer-coated glass square (1 mm
thick; 25 mm square). A second cleaned, polymer-coated glass square
was aligned with the first square and pressure applied to uniformly
distribute the sample. A sample depth of approximately 20 .mu.m was
maintained by mylar spacers located between the two glass squares.
The edges of the glass assay chambers were sealed with nail
polish.
[0089] Liquid crystals are anisometric molecules that exhibit
limited chemical interaction but that tend to orient along a common
direction (the director). Director orientation is affected by
externally applied fields (electrical and magnetic); at the
boundary between the liquid crystal and it's container and flow.
The liquid crystal orientation was optimized by constructing glass
assay chambers that enhanced container-liquid crystal interaction.
The chambers were created as follows: Borosilicate glass (1.0 mm
thick; 200 mm.times.200 mm) plates were cleaned for 5 minutes in an
60.degree. C., ultrasonic bath containing Alconox Detergent (Fisher
Scientific; Hanover Park, Ill.; product # 04-322-4) in water,
rinsed in distilled water and dried at 100.degree. C. Each plate
was coated with an aligned layer of a polymer. The glass plate was
cut into 25 mm squares. A 25 mm square was positioned polymer up
with two mylar spacer strips (20 .mu.m thick, 2.0 mm.times.25 mm)
located on the outer edges of the glass parallel to the orientation
of the polymer. Liquid crystal-microsphere samples (60 .mu.l) were
applied at the bottom edge of the glass between the mylar strips
and a second 25 mm polymer-coated glass was positioned so that it's
polymer orientation was parallel to the bottom glass. Pressure was
applied to the top glass to distribute the sample. The edges were
sealed with an appropriate sealing material.
[0090] Two liquid crystal solutions were evaluated. Lyotropic
liquid crystals were formed when either 20% disodium cromoglycate
(Hartshorne and Woodard, Mol. Cryst. Liq. Cryst. 23:343, 1973) or
20% neutral grey was added to 80% distilled water (w/v).
Preliminary phase diagrams demonstrate that both the disodium
cromoglycate (Sigma Chem. Co, St. Louis, Mo.; product # C0399) and
the neutral grey (Optiva Inc., San Francisco, Calif.) liquid
crystalline solutions remained in nematic phase at 24.degree. C.
when diluted to a 14% solution.
[0091] For Examples 1-8, cultures of E. coli (ATCC number 23503),
grown to mid log growth phase in tryptic soy broth (Becton
Dickinson, Sparks, Md.; product # 211822), were washed free of
growth medium with two washes of Phosphate Buffered Saline were
used. The optical density of each E. coli suspension at 600 nm was
measured and the bacteria concentration extrapolated from a growth
curve (optical density at 600 nm versus colony-forming units
(CFUs)). Bacteria were then diluted with sterile phosphate buffered
saline (PBS) to a concentration of 10.sup.8 CFU per 10 .mu.l.
[0092] Each mixture was evaluated for light transmissive zones at
200.times.magnification using a microscope equipped with crossed
polarizers. For each assay cassette, the number of light
transmissive zones in ten microscope fields were counted and the
mean number per field calculated. Each experiment was conducted in
duplicate. The data points in each of the following graphs
represent the mean of the duplicate experiments.
Example 1
[0093] A commercially available 1.0 .mu.m diameter polystyrene
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polystyrene microsphere was coated with a protein that tightly
binds microbe specific antibodies. Protein G, a S. aureus protein
that binds the Fc fraction of immunoglobulins, was cross-linked to
the outer surface of the polystyrene microspheres.
[0094] A commercially available murine antibody (Accurate Chemical
Co.; Westbury, N.Y.; product # YCC-311-603) specific to the sex
pili (K99) of E. coli bacteria was obtained and used undiluted. A
stock solution of assay microspheres (10.sup.7/.mu.l) was created
by incubating 44 .mu.l of microspheres with 56 .mu.l of the murine
anti-E. coli antibody for 30 minutes at room temperature. The
solution was washed twice with phosphate buffered saline to remove
unbound primary antibody.
[0095] 10 .mu.l of serially diluted polystyrene microspheres coated
with the anti-E. coli K99 antibody was mixed with 10 .mu.l of the
stock E. coli solution and incubated for 30 minutes at room
temperature. The 20% stock solution of neutral grey liquid crystal
(50 .mu.l) was added to the microsphere-antibody solution, mixed,
and the samples gently centrifuged (3500 g; 5 sec.) to eliminate
bubbles. A 60 .mu.l fraction of the mixture was introduced into the
glass assay chamber described above.
Comparative Example 2
[0096] A commercially available 1.0 .mu.m diameter polystyrene
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polystyrene microsphere was coated with a protein that tightly
binds microbe specific antibodies. Protein G, a S. aureus protein
that binds the Fc fraction of immunoglobulins, was cross-linked to
the outer surface of the polystyrene microspheres.
[0097] A stock solution of assay microspheres (10.sup.7/.mu.l) was
created by incubating 44 .mu.l of microspheres with 56 .mu.l BSA
for 30 minutes at room temperature. The solution was washed twice
with phosphate buffered saline to remove unbound primary
antibody.
[0098] 10 .mu.l of serially diluted polystyrene microspheres coated
with BSA was mixed with 10 .mu.l of the stock E. coli solution and
incubated for 30 minutes at room temperature. The 20% stock
solution of the neutral grey liquid crystal (50 .mu.l) was added to
the microsphere-antibody solution, mixed, and the samples gently
centrifuged (3500 g; 5 sec.) to eliminate bubbles. A 60 .mu.l
fraction of the mixture was introduced into the glass assay chamber
described above.
Example 3
[0099] A commercially available 1.0 .mu.m diameter polycarboxylate
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polycarboxylate microsphere was coated with a protein that tightly
binds microbe specific antibodies. The polycarboxylate microsphere
was coated with a goat immunoglobulin that binds all mouse
immunoglobulins.
[0100] As described in Example 1 above, a commercially available
murine antibody specific to the sex pili (K99) of E. coli bacteria
was obtained and used undiluted. A stock solution of assay
microspheres (10.sup.7/.mu.l) was created by incubating 44 .mu.l of
microspheres with 56 .mu.l of the murine anti-E. coli antibody for
30 minutes at room temperature. The solution was washed twice with
phosphate buffered saline to remove unbound primary antibody.
[0101] 10 .mu.l of serially diluted polycarboxylate microspheres
coated with the anti-E. coli K99 antibody was mixed with 10 .mu.l
of the stock E. coli solution and incubated for 30 minutes at room
temperature. The 20% stock solution of neutral grey liquid crystal
(50 .mu.l) was added to the microsphere-antibody solution, mixed,
and the samples gently centrifuged (3500 g; 5 sec.) to eliminate
bubbles. A 60 .mu.l fraction of the mixture was introduced into the
glass assay chamber described above.
Comparative Example 4
[0102] A commercially available 1.0 .mu.m diameter polycarboxylate
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polycarboxylate microsphere was coated with a protein that tightly
binds microbe specific antibodies. The polycarboxylate microsphere
was coated with a goat immunoglobulin that binds all mouse
immunoglobulins.
[0103] A stock solution of assay microspheres (10.sup.7/.mu.l) was
created by incubating 44 .mu.l of microspheres with 56 .mu.l of BSA
for 30 minutes at room temperature. The solution was washed twice
with phosphate buffered saline to remove unbound primary
antibody.
[0104] 10 .mu.l of serially diluted polycarboxylate microspheres
coated with BSA was mixed with 10 /.mu.l of the stock E. coli
solution and incubated for 30 minutes at room temperature. The 20%
stock solution of the neutral grey liquid crystal (50 .mu.l) was
added to the microsphere-antibody solution, mixed, and the samples
gently centrifuged (3500 g; 5 sec.) to eliminate bubbles. A 60
.mu.l fraction of the mixture was introduced into the glass assay
chamber described above.
[0105] Example 5
[0106] A commercially available 1.0 .mu.m diameter polystyrene
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polystyrene microsphere was coated with a protein that tightly
binds microbe specific antibodies. Protein G, a S. aureus protein
that binds the Fc fraction of immunoglobulins, was cross-linked to
the outer surface of the polystyrene microspheres.
[0107] A commercially available murine antibody (Accurate Chemical
Co.; Westbury, N.Y.; product # YCC-311-603) specific to the sex
pili (K99) of E. coli bacteria was obtained and used undiluted. A
stock solution of assay microspheres (10.sup.7/.mu.l) was created
by incubating 44 .mu.l of microspheres with 56 .mu.l of the murine
anti-E. coli antibody for 30 minutes at room temperature. The
solution was washed twice with phosphate buffered saline to remove
unbound primary antibody.
[0108] 10 .mu.l of serially diluted polystyrene microspheres coated
with the anti-E. coli K99 antibody was mixed with 10 .mu.l of the
stock E. coli solution and incubated for 30 minutes at room
temperature. The 20% stock solution of disodium cromoglycate liquid
crystal (50 .mu.L) was added to the microsphere-antibody solution,
mixed, and the samples gently centrifuged (3500 g; 5 sec.) to
eliminate bubbles. A 60 .mu.l fraction of the mixture was
introduced into the glass assay chamber described above.
[0109] Comparative Example 6
[0110] A commercially available 1.0 .mu.m diameter polystyrene
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polystyrene microsphere was coated with a protein that tightly
binds microbe specific antibodies. Protein G, a S. aureus protein
that binds the Fc fraction of immunoglobulins, was cross-linked to
the outer surface of the polystyrene microspheres.
[0111] A stock solution of assay microspheres (10.sup.7/.mu.l) was
created by incubating 44 .mu.l of microspheres with 56 .mu.l BSA
for 30 minutes at room temperature. The solution was washed twice
with phosphate buffered saline to remove unbound primary
antibody.
[0112] 10 .mu.l of serially diluted polystyrene microspheres coated
with BSA was mixed with 10 .mu.l of the stock E. coli solution and
incubated for 30 minutes at room temperature. The 20% stock
solution of the disodium cromoglycate liquid crystal (50 .mu.l) was
added to the microsphere-antibody solution, mixed, and the samples
gently centrifuged (3500 g; 5 sec.) to eliminate bubbles. A 60
.mu.l fraction of the mixture was introduced into the glass assay
chamber described above.
[0113] Example 7
[0114] A commercially available 1.0 .mu.m diameter polycarboxylate
microsphere was obtained (Polysciences, Inc, Warrington, Pa.). The
polycarboxylate microsphere was coated with a protein that tightly
binds microbe specific antibodies. The polycarboxylate microsphere
was coated with a goat immunoglobulin that binds all mouse
immunoglobulins.
[0115] As described in Example 1 above, a commercially available
murine antibody specific to the sex pili (K99) of E. coli bacteria
was obtained and used undiluted. A stock solution of assay
microspheres (10.sup.7/.mu.l) was created by incubating 44 .mu.l of
microspheres with 56 .mu.l of the murine anti-E. coli antibody for
30 minutes at room temperature. The solution was washed twice with
phosphate buffered saline to remove unbound primary antibody.
[0116] 10 .mu.l of serially diluted polycarboxylate microspheres
coated with the anti-E. coli K99 antibody was mixed with 10 .mu.l
of the stock E. coli solution and incubated for 30 minutes at room
temperature. The 20% stock solution of disodium cromoglycate liquid
crystal (50 .mu.l) was added to the microsphere-antibody solution,
mixed, and the samples gently centrifuged (3500 g; 5 sec.) to
eliminate bubbles. A 60 .mu.l fraction of the mixture was
introduced into the glass assay chamber described above.
Comparative Example 8
[0117] A commercially available 1.0 .mu.m diameter polycarboxylate
microsphere was obtained (Polysciences, Inc, Warrington, Pa). The
polycarboxylate microsphere was coated with a protein that tightly
binds microbe specific antibodies. The polycarboxylate microsphere
was coated with a goat immunoglobulin that binds all mouse
immunoglobulins.
[0118] A stock solution of assay microspheres (10.sup.7/.mu.l) was
created by incubating 44 .mu.l of microspheres with 56 .mu.l of BSA
for 30 minutes at room temperature. The solution was washed twice
with phosphate buffered saline to remove unbound primary
antibody.
[0119] 10 .mu.l of serially diluted polycarboxylate microspheres
coated with BSA was mixed with 10 .mu.l of the stock E. coli
solution and incubated for 30 minutes at room temperature. The 20%
stock solution of the disodium chromoglycate liquid crystal (50
.mu.l) was added to the microsphere-antibody solution, mixed, and
the samples gently centrifuged (3500 g; 5 sec.) to eliminate
bubbles. A 60 .mu.l fraction of the mixture was introduced into the
glass assay chamber described above.
[0120] Ligand (bacteria)-bound microsphere aggregates distorted the
liquid crystal director to cause local zones of light transmission,
which were easily detected.
[0121] FIG. 4A demonstrates that increasing numbers of light
transmissive zones occur in a 14% neutral grey liquid crystalline
solution as the ratio of E. coli to polycarboxylate microspheres
increases.
[0122] FIG. 4B demonstrates that increasing numbers of light
transmissive zones occur in a 14% neutral grey liquid crystalline
solution as the ratio of E. coli to polystyrene microspheres
increases.
[0123] FIG. 5A shows that increasing numbers of light transmissive
zones occur in a 14% disodium cromoglycate liquid crystalline
solution as the ratio of E. coli to polycarboxylate microspheres
increases.
[0124] FIG. 5B shows that increasing numbers of light transmissive
zones occur in a 14% disodium cromoglycate liquid crystalline
solution as the ratio of E. coli to polystyrene microspheres
increases.
[0125] Greater light transmission occurred at microsphere to E.
coli ratios exceeding 1:4. In all experiments, antibody-coated
microspheres induced the formation of more light transmissive zones
than did the control microspheres coated with Bovine Serum
Albumin.
[0126] It is to be understood that any variations evident fall
within the scope of the claimed invention, and thus the selection
of specific receptors, such as antibodies and liquid crystals can
be determined without departing from the spirit of the invention
herein disclosed and described. It should also be understood that
the present invention, while particularly suited for pathogen
detection, is intended to include the detection of any ligand.
Moreover, the scope of the invention shall include all
modifications and variations that may fall within the scope of the
attached claims.
* * * * *