U.S. patent application number 10/997439 was filed with the patent office on 2005-07-07 for very large scale immobilized polymer synthesis.
This patent application is currently assigned to Affymetrix Inc.. Invention is credited to Fodor, Stephen P.A., Pirrung, Michael C., Read, J. Leighton, Stryer, Lubert.
Application Number | 20050148027 10/997439 |
Document ID | / |
Family ID | 27001838 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050148027 |
Kind Code |
A1 |
Pirrung, Michael C. ; et
al. |
July 7, 2005 |
Very large scale immobilized polymer synthesis
Abstract
A method and apparatus for preparation of a substrate containing
a plurality of sequences. Photoremovable groups are attached to a
surface of a substrate. Selected regions of the substrate are
exposed to light so as to activate the selected areas. A monomer,
also containing a photoremovable group, is provided to the
substrate to bind at the selected areas. The process is repeated
using a variety of monomers such as amino acids until sequences of
a desired length are obtained. Detection methods and apparatus are
also disclosed.
Inventors: |
Pirrung, Michael C.;
(Durham, NC) ; Read, J. Leighton; (Palo Alto,
CA) ; Fodor, Stephen P.A.; (Palo Alto, CA) ;
Stryer, Lubert; (Stanford, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW LLP
TWO EMBARCADERO CENTER
8TH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Affymetrix Inc.
Santa Clara
CA
|
Family ID: |
27001838 |
Appl. No.: |
10/997439 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10997439 |
Nov 23, 2004 |
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10428628 |
May 2, 2003 |
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10428628 |
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Mar 15, 2002 |
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Oct 16, 2000 |
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Current U.S.
Class: |
435/7.1 ;
257/E21.705; 435/287.2; 436/518; 530/333 |
Current CPC
Class: |
B01J 2219/00626
20130101; B82Y 30/00 20130101; C40B 40/06 20130101; B01J 2219/00619
20130101; C07K 1/062 20130101; G11C 13/0019 20130101; B01J
2219/00468 20130101; B01J 2219/00585 20130101; B01J 2219/00621
20130101; C07B 2200/11 20130101; C12Q 1/6874 20130101; B01J
2219/0061 20130101; Y10T 428/31971 20150401; B01J 2219/00434
20130101; B01J 2219/00711 20130101; C07K 1/04 20130101; C12Q 1/6816
20130101; B01J 2219/00605 20130101; C40B 40/10 20130101; B01J
2219/00529 20130101; B01J 2219/00617 20130101; B01J 2219/00659
20130101; C07C 229/14 20130101; C07H 19/04 20130101; C07K 1/047
20130101; G03F 7/00 20130101; B01J 2219/00459 20130101; B01J
2219/00608 20130101; B01J 2219/00527 20130101; C07K 17/14 20130101;
B01J 2219/00648 20130101; C07K 17/06 20130101; Y10S 435/961
20130101; B01J 2219/00637 20130101; C07H 21/00 20130101; C07K 7/06
20130101; C07C 229/16 20130101; G01N 21/6428 20130101; B01J
2219/00436 20130101; B01J 2219/005 20130101; G11C 13/0014 20130101;
B01J 2219/00639 20130101; G01N 21/253 20130101; B01J 2219/00315
20130101; B82Y 10/00 20130101; C07H 19/10 20130101; G01N 15/1475
20130101; Y10S 436/807 20130101; Y10S 436/809 20130101; C12Q 1/6837
20130101; C07D 263/44 20130101; C07K 1/042 20130101; C12Q 1/6809
20130101; G03F 7/26 20130101; B01J 2219/00432 20130101; C07D 317/62
20130101; C08G 69/00 20130101; Y10S 435/973 20130101; Y10T 436/2575
20150115; C40B 80/00 20130101; H01L 25/50 20130101; Y10S 435/968
20130101; B01J 2219/0059 20130101; B01J 2219/00689 20130101; B01L
7/52 20130101; B01J 2219/00596 20130101; B01J 2219/00612 20130101;
Y02P 20/55 20151101; B01J 2219/00475 20130101; B01J 2219/00531
20130101; C07K 1/045 20130101; G03F 7/265 20130101; B01J 19/0046
20130101; B01J 2219/00695 20130101; C40B 60/14 20130101; Y10S
435/969 20130101; B01J 2219/00722 20130101; G03F 7/38 20130101;
H01L 2924/0002 20130101; B01J 2219/00725 20130101; G01N 21/6452
20130101; H01L 2924/0002 20130101; H01L 2924/00 20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2; 436/518; 530/333 |
International
Class: |
C12Q 001/68; G01N
033/53; C12M 001/34; C07K 001/02 |
Claims
What is claimed is:
1. A method of preparing sequences on a substrate comprising the
steps of: a) exposing a first region of said substrate to an
activator to remove a protective group; b) exposing at least said
first region to a first monomer; c) exposing a second region to an
activator to remove a protective group; and d) exposing at least
said second region to a second monomer.
2. The method as recited in claim 1 wherein said steps of exposing
to an activator use an activator selected from the group consisting
of ion beams, electron beams, gamma rays, x-rays, ultra-violet
radiation, light, infra-red radiation, microwaves, electric
currents, radiowaves, and combinations thereof.
3. The method as recited in claim 1 wherein said protective groups
are photosensitive protective groups.
4. The method as recited in claim 1 wherein said steps of exposing
to an activator are steps of applying light to selected regions of
said substrate.
5. The method as recited in claim 1 wherein said first and the
second monomers are amino acids.
6. The method as recited in claim 1 further comprising a step of
screening sequences on said substrate for affinity with a receptor,
said step of screening further comprising the step of exposing said
substrate to said receptor and testing for the presence of said
receptor in said first and said second region.
7. The method as recited in claim 6 wherein said receptor is an
antibody.
8. The method as recited in claim 1 wherein said substrate is
selected from the group consisting of polymerized Langmuir Blodgett
film, functionalized glass, germanium, silicon, polymers,
(poly)tetrafluoroethylene, polystyrene, gallium arsenide, and
combinations thereof.
9. The method as recited in claim 1 wherein said protective group
is selected from the group consisting of ortho-nitrobenzyl
derivatives, 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl,
cinnamoyl derivatives, and mixtures thereof.
10. The method as recited in claim 1 wherein said first and second
regions each have total areas of less than 1 cm.sup.2.
11. The method as recited in claim 1 wherein said first and second
regions each have total areas of between about 1 .mu.m.sup.2 and
10,000 .mu.m.sup.2.
12. The method as recited in claim 4 wherein said light is
monochromatic coherent light.
13. The method as recited in claim 1 wherein said steps of exposing
to an activator are carried out with a solution in contact with
said substrate.
14. The method as recited in claim 13 wherein said solution further
comprises said first or said second monomer.
15. The method as recited in claim 6 wherein said receptor further
comprises a marker selected from the group consisting of
radioactive markers and fluorescent markers and wherein said step
of testing for the presence of the receptor is a step of detecting
said marker.
16. The method as recited in claim 1 wherein the steps of exposing
to an activator further comprise steps of: a) placing a mask
adjacent to said substrate, said mask having substantially
transparent regions and substantially opaque regions at a
wavelength of light; and b) illuminating said mask with a light
source, said light source producing at least said wavelength of
light.
17. The method as recited in claim 1 wherein said steps are
repeated so as to synthesize 10.sup.3 or more different sequences
on said substrate.
18. The method as recited in claim 1 wherein said steps are
repeated so as to synthesize 10.sup.6 or more different sequences
on said substrate.
19. A method of synthesizing a plurality of chemical sequences,
said chemical sequences comprising at least a first and a second
monomer, comprising the steps of: a) at a first region on a
substrate having at least a first and a second region, said first
and said second region comprising a substrate protective group,
activating said first region to remove said substrate protective
group in said first region; b) exposing said first monomer to said
substrate, said first monomer further comprising a first monomer
protective group, said first monomer binding at said first region;
c) activating said second region to remove said substrate
protective group in said second region; d) exposing said second
monomer to said substrate, said second monomer further comprising a
second monomer protective group, said second monomer binding at
said second region; e) activating said first region to remove said
first monomer protective group; f) exposing a third monomer to said
substrate, said third monomer binding at said first region to
produce a first sequence; g) activating said second region to
remove said second monomer protective group; and h) exposing a
fourth monomer to said substrate, said fourth monomer binding at
said second region to produce a second sequence, said second
sequence different from said first sequence.
20. A method of synthesizing a plurality of chemical sequences,
said chemical sequences comprising at least a first and a second
monomer, comprising the steps of: a) on a substrate having at least
a first and a second region deactivating said first region to
provide a first protective group in said first region; b) exposing
said first monomer to said substrate, said first monomer binding at
said second region; c) removing said protective group in said first
region; d) deactivating said second region to provide a second
protective group in said second region; e) exposing said second
monomer to said substrate, said second monomer binding at said
first region; f) removing said protective group in said second
region; g) deactivating said first region to provide a protective
group in said first region; h) exposing a third monomer to said
substrate, said third monomer binding at said second region to
produce a first sequence; i) removing said protective group in said
first region; and j) exposing a fourth monomer to said substrate,
said fourth monomer binding at said first region to produce a
second sequence, said second sequence different than said first
sequence.
21. A method of synthesizing at least a first polymer sequence and
a second polymer sequence on a substrate, said first polymer
sequence having a different monomer sequence from said second
polymer sequence, comprising the steps of: a) inserting a first
mask between said substrate and an energy source, said mask having
first regions and second regions, said first regions permitting
passage of energy from said source, said second regions blocking
energy from said source; b) directing energy from said source at
said substrate, said energy removing a protective group from first
portions of said first polymer under said first regions of said
first mask; c) exposing a second portion of said first polymer to
said substrate to create a first polymer sequence; d) inserting a
second mask between said substrate and said energy source, said
second mask having first regions and second regions; e) directing
energy from said source at said substrate, said energy removing
said protective group under said first regions of said second mask
from first portions of said second polymer; and f) exposing a
second portion of said second polymer to said substrate, said
second portion of said second polymer binding with said first
portion of said second polymer to create a polymer 8 second
sequence.
22. The method as recited in claim 21 wherein said energy is
selected from the group consisting of ion beams, electron beams,
gamma rays, x-rays, ultra-violet radiation, light, infra-red
radiation, microwaves, electric fields, radiowaves, and
combinations thereof.
23. The method as recited in claim 19 wherein said protective
groups are photosensitive protective groups.
24. The method as recited in claims 19 or 20 wherein said steps of
activating and deactivating are steps of applying light to selected
regions of said substrate.
25. The method as recited in claims 19 or 20 wherein said first and
said second monomers are amino acids.
26. The method as recited in claims 19, 20 or 21 further comprising
a step of screening said first and said second sequences for
affinity with a first receptor, said step of screening further
comprising a step of exposing said substrate to said first receptor
and testing for the presence of said first receptor.
27. The method as recited in claim 26 wherein said step of
screening is a step of screening with antibodies.
28. The method as recited in claims 19, 20 or 21 wherein said
substrate is selected from the group consisting of a polymerized
Langmuir Blodgett film, functionalized glass, germanium, silicon,
polymers, (poly)tetrafluoroethylene, gallium arsenide, gallium
phosphide, silicon oxide, silicon nitride and combinations
thereof.
29. The method as recited in claim 19 wherein said protective
group, said first monomer protective group, and said second monomer
protective group are selected from the group consisting of
ortho-nitrobenzyl derivatives, 6-nitroveratryloxycarbonyl,
2-nitrobenzyloxycarbonyl, and mixtures thereof.
30. The method as recited in claim 20 wherein said protective group
is a cinnamate group.
31. The method as recited in claims 19 or 20 wherein said first and
second regions each have total areas of less than 1 cm.sup.2.
32. The method as recited in claims 19 or 20 wherein said first and
second regions each have total areas of between about 1 .mu.m.sup.2
and 10,000 .mu.m.sup.2.
33. The method as recited in claim 24 wherein said light is
monochromatic coherent light.
34. The method as recited in claim 19 wherein said steps of
activating are carried out with a solution in contact with said
substrate.
35. The method as recited in claim 34 wherein said solution further
comprises a monomer.
36. The method as recited in claim 26 wherein said receptor further
comprises a marker selected from the group consisting of
radioactive markers and fluorescent markers and wherein said step
of testing for the presence of the receptor is a step of detecting
said marker.
37. The method as recited in claims 19 or 20 wherein two of said
first, said second, said third, and said fourth monomers are the
same monomers.
38. The method as recited in claim 21 wherein the step of inserting
a second mask is a step of translating said first mask from a first
position to a second position.
39. The method as recited in claim 21 wherein the step of inserting
a second mask is a step of rotating said first mask.
40. The method as recited in claim 26 further comprising the step
of exposing said substrate to a second, labeled receptor, said
second, labeled receptor binding at multiple sites on said first
receptor.
41. The method as recited in claim 40 wherein said first receptor
is an antibody of a first animal species and said second receptor
is an antibody derived from a second species and directed at said
first species.
42. The method as recited in claim 19 wherein: a) said first
monomer protective group is removable upon exposure to a first
wavelength of light; b) said second monomer protective group is
removable upon exposure to a second wavelength of light; c) said
step of activating said first region to remove said first monomer
protective group is a step of exposing substantially all of said
substrate to said first wavelength of light; and d) said step of
activating said second region to remove said second monomer
protective group is a step of exposing substantially all of said
substrate to said second wavelength of light.
43. A method as recited in claims 19 or 21 wherein said protective
groups are of the form: 2where R.sub.1 is alkoxy, alkyl, halo,
aryl, alkenyl, or hydrogen; R.sub.2 is alkoxy, alkyl, halo, aryl,
nitro, or hydrogen; R.sub.3 is alkoxy, alkyl, halo, nitro, aryl, or
hydrogen; R.sub.4 is alkoxy, alkyl, hydrogen, aryl, halo, or nitro;
and R.sub.5 is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo, aryl,
or alkenyl.
44. A method of screening a plurality of amino acid sequences for
binding with a receptor comprising the steps of: a) on a glass
plate having at least a first surface, said at least a first
surface comprising a photoprotective material selected from the
group consisting of nitroveratryloxy carbonyl and nitrobenzyloxy
carbonyl, reacting said at least a first surface with
t-butoxycarbonyl for storage, said glass plate substantially
transparent to at least ultraviolet light; b) exposing said at
least a first surface to TFA to remove said t-butoxycarbonyl; c)
placing said glass plate on a reactor, said reactor comprising a
reactor space, said at least a first surface exposed to said
reactor space; d) placing a mask at a first position on said glass
plate, said mask comprising first locations and second locations,
said first locations substantially transparent to at least
ultraviolet light and said second locations substantially opaque to
at least ultraviolet light, said second locations comprising a
light blocking material on a first surface of said mask, said first
surface of said mask placed in contact with said glass plate; e)
filling said reactor space with a reaction solution; f)
illuminating said mask with at least ultraviolet light, said
ultraviolet light removing said photoprotective material from said
at least a first surface of said glass plate under said first
locations of said mask; g) exposing said first surface to a first
amino acid, said first amino acid binding to regions of said at
least a first surface from which said photoprotective material was
removed, said first amino acid comprising said photoprotective
group at a terminus thereof; h) placing a mask in contact with said
glass plate at a second position; i) illuminating said mask with at
least ultraviolet light, said ultraviolet light removing said
photoprotective material from said at least a first surface of said
glass plate under said first locations of said mask; j) exposing
said at least a first surface to a second amino acid, said second
amino acid binding to regions of said at least a first surface from
which said photoprotective material was removed, said second amino
acid comprising said photoprotective group at a terminus thereof;
k) placing a mask in contact with said glass plate at a third
position; l) illuminating said mask with at least ultraviolet
light, said ultraviolet light removing said photoprotective
material from said at least a first surface of said glass plate
under said first locations of said mask; m) exposing said at least
a first surface to a third amino acid, said third amino acid
binding to regions of said at least a first surface from which said
photoprotective material was removed; n) placing a mask in contact
with said glass plate at a fourth position; o) illuminating said
mask with at least ultraviolet light, said ultraviolet light
removing said photoprotective material from said at least a first
surface of said glass plate under said first locations of said
mask; p) exposing said at least a first surface to a fourth amino
acid, said fourth amino acid binding to regions of said at least a
first surface from which said photoprotective material was removed,
said at least a first surface comprising at least first, second,
third, and fourth amino acid sequences; q) exposing said at least a
first surface to an antibody of interest, said antibody of interest
binding more strongly to at least one of said first, said second,
said third, or said fourth amino acid sequences; r) exposing said
at least a first surface to a receptor, said receptor recognizing
said antibody of interest and binding at multiple locations
thereof, said receptor comprising fluorescein; s) exposing said at
least a first surface to light, said first surface fluorescing in
at least a region where said more strongly bound amino acid
sequence is located; and t) detecting and recording fluoresced
light intensity as a function of location across said at least a
first surface.
45. A method of identifying at least one peptide sequence for
binding with a receptor comprising the steps of: a) on a substrate
having a plurality of polypeptides, each having a photoremovable
protective group, irradiating first selected polypeptides to remove
said protective group; b) contacting said polypeptides with a first
amino acid to create a first sequence, second polypeptides on said
substrate comprising a second sequence; and c) identifying which of
said first or said second sequence binds with said receptor.
46. The method as recited in claim 45 wherein said step of
identifying further comprises a step of detecting the presence of a
marker selected from the group consisting of radioactive markers
and fluorescent markers in said receptor.
47. The method as recited in claim 45 wherein said step of
irradiating is a step of masking a light source with a mask, said
mask comprising first transparent regions and second opaque
regions.
48. The method as recited in claim 47 wherein the step of
identifying further comprises the steps of: a) exposing a first
receptor to said substrate; and b) exposing a receptor to said
first receptor to said substrate, said receptor to said first
receptor comprising a marker.
49. The method as recited in claim 48 wherein said marker is
selected from the group consisting of radioactive markers and
fluorescent markers.
50. The method as recited in claim 48 wherein said first receptor
is an antibody from a first species and said receptor to said first
receptor is an antibody from a second species directed at said
first species.
51. A method for screening a plurality of polymers for biological
activity comprising exposing a receptor to a substrate having said
plurality of said polymers on a surface thereof, each of said
polymers occupying an area of less than about 1 cm.sup.2.
52. A method for screening as recited in claim 48 wherein said area
is less than about 0.1 cm.sup.2.
53. A method as recited in claim 48 wherein said area is less than
about 10,000 .mu.m.sup.2.
54. A method as recited in claim 48 wherein said area is less than
about 100 .mu.m.sup.2.
55. Apparatus for preparation of a plurality of polymers
comprising: a) a substrate with a surface, said surface comprising
a reactive portion, said reactive portion activated upon exposure
to an energy source so as to react with a monomer; and b) means for
selectively protecting and exposing portions of said surface from
said energy source.
56. Apparatus as recited in claim 55 wherein said reactive portion
further comprises a protective group, said protective group of the
form: 3where R.sub.1 is alkoxy, alkyl, halo, aryl, alkenyl, or
hydrogen; R.sub.2 is alkoxy, alkyl, halo, aryl, nitro, or hydrogen;
R.sub.3 is alkoxy, alkyl, halo, nitro, aryl, or hydrogen; R.sub.4
is alkoxy, alkyl, hydrogen, aryl, halo, or nitro; and R.sub.5 is
alkyl, alkynyl, cyano, alkoxy, hydrogen, halo, aryl, or
alkenyl.
57. Apparatus as recited in claim 55 wherein said reactive portion
further comprises linker molecules.
58. Apparatus as recited in claim 57 wherein said linker molecules
are selected from the group consisting of ethylene glycol
oligomers, diamines, diacids, amino acids, and combinations
thereof.
59. Apparatus as recited in claim 55 wherein said means for
selectively protecting further comprises a mask.
60. Apparatus as recited in claim 55 wherein said means for
selectively protecting further comprises a light valve.
61. Apparatus as recited in claim 55 wherein said energy source is
a light source.
62. Apparatus as recited in claim 55 wherein said reactive portion
further comprises a composition selected from the group consisting
of nitroveratryloxy carbonyl, nitrobenzyloxy carbonyl,
dimethyl-dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl,
hydroxy-2-methyl cinnamoyl, and 2-oxymethylene anthraquinone.
63. Apparatus for preparation of a substrate having a plurality of
amino acid sequences thereon, said apparatus comprising: a) a
substrate with a surface; b) a protective group on said surface,
said protective group removable upon exposure to an energy source,
said energy source selected from the group consisting of light,
electron beams, and x-ray radiation; c) means for directing said
energy source at selected locations on said surface; and d) means
for exposing amino acids to said surface for binding to said
surface.
64. Apparatus for screening polymers comprising a substrate with a
surface, said surface comprising at least two predefined regions,
said predefined regions containing different monomer sequences
thereon, said predefined regions each occupying an area of less
than about 0.1 cm.sup.2.
65. Apparatus as recited in claim 64 wherein said area is less than
about 0.01 cm.sup.2.
66. Apparatus as recited in claim 64 wherein said area is less than
10000 .mu.m.sup.2.
67. Apparatus as recited in claim 64 wherein said area is less than
about 100 .mu.m.sup.2.
68. Apparatus as recited in claims 64, 65, 66, or 67 wherein said
monomer sequences are substantially pure within said predefined
regions.
69. A substrate for screening for biological activity, said
substrate comprising 10.sup.3 or more different ligands on a
surface thereof in predefined regions.
70. A substrate as recited in claim 69 wherein said substrate
comprises 10.sup.4 or more different ligands in predefined
regions.
71. A substrate as recited in claim 69 wherein said substrate
comprises 10.sup.5 or more different ligands in predefined
regions.
72. A substrate as recited in claim 69 wherein said substrate
comprises 10.sup.6 or more different ligands in predefined
regions.
73. A substrate as recited in claims 69, 70, 71, or 72 wherein the
ligands are peptides.
74. A substrate as recited in claim 64 wherein said ligands are
substantially pure within said predefined regions.
75. Apparatus for screening for biological activity comprising: a)
a substrate comprising a plurality of polymer sequences, said
polymer sequences attached to a surface of said substrate at known
locations on said substrate, each of said sequences occupying an
area of less than about 0.1 cm.sup.2; b) means for exposing said
substrate to a receptor, said receptor marked with a fluorescent
marker, said receptor binding with at least one of said sequences;
and c) means for detecting a location of said fluorescent marker on
said substrate.
76. Apparatus for forming a plurality of polymer sequences
comprising: a) a substrate, said substrate having at least a first
surface and a second surface, said second surface comprising a
photoremovable protective material, said substrate substantially
transparent to at least light of a first wavelength; b) a reactor
body, said reactor body having a mounting surface with a reaction
fluid cavity therein, said second surface maintained in a sealed
relationship with said mounting surface; and c) a light source for
producing light of at least said first-wavelength and directed at a
surface of said substrate.
77. Apparatus as recited in claim 76 wherein said light source is
directed at said first surface.
78. Apparatus as recited in claim 76 further comprising a mask,
said mask placed between said light source and said first surface,
said mask having first regions substantially transparent to said
first wavelength of light and second regions substantially opaque
to said first wavelength of light.
79. Apparatus as recited in claim 76 wherein said cavity comprises
a fluid inlet and a fluid outlet, said fluid inlet connected to a
pump for flowing reaction fluids through said cavity.
80. Apparatus as recited in claim 76 wherein said cavity further
comprises a plurality of raised sections.
81. Apparatus as recited in claim 78 wherein said mask further
comprises a glass plate.
82. Apparatus as recited in claim 81 wherein said opaque regions on
said mask comprise chrome.
83. Apparatus as recited in claim 76 wherein at least a portion of
said second surface comprises a second photoremovable protective
group, said second photoremovable protective group activatable upon
exposure to light of a second wavelength.
84. Apparatus as recited in claim 76 further comprising first and
second gaskets on said mounting surface and means for maintaining a
vacuum between said first and second gaskets.
85. Apparatus as recited in claim 76 wherein said substrate has a
thickness of less than 1 mm.
86. Apparatus as recited in claim 76 wherein said substrate has a
thickness of less than 0.5 mm.
87. Apparatus as recited in claim 76 wherein said substrate has a
thickness of less than 0.05 mm.
88. Apparatus as recited in claim 78 wherein said mask is in direct
contact with said substrate.
89. Apparatus as recited in claim 88 wherein opaque regions of said
mask are placed in direct contact with said substrate.
90. Apparatus as recited in claim 76 further comprising a liquid
crystal light valve for selectively controlling exposure of light
to said substrate.
91. Apparatus as recited in claim 76 further comprising a fiber
optic faceplate between said light source and said substrate.
92. Apparatus as recited in claim 76 further comprising a molecular
microcrystal between said light source and said substrate.
93. Apparatus as recited in claim 76 wherein said cavity comprises
light absorptive materials.
94. Apparatus as recited in claim 93 wherein said light absorptive
material is N,N-diethylamino 2,4-dinitrobenzene.
95. Apparatus as recited in claim 76 wherein said cavity is filled
with a carrier solution.
96. Apparatus as recited in claim 95 wherein said carrier material
comprises a material selected from the group of
1-hydroxybenzotriazole, dimethylformamide, diisopropylethylamine,
and benzotriazolyl-n-oxy-tris (dimethylamino)
phosphoriumhexafluorophosphate.
97. Apparatus as recited in claim 76 wherein said substrate is a
fiber optic faceplate.
98. Apparatus for detection of fluorescent marked regions on a
substrate comprising: a) a light source for directing light at a
surface of said substrate; b) a means for detecting light
fluoresced from said surface in response to said light source; c)
means for translating said substrate from a first position to a
second position; and d) means for storing fluoresced light
intensity as a function of location on said substrate, said means
for storing connected to said means for translating and said means
for detecting.
99. Apparatus as recited in claim 98 further comprising video
display means for displaying light intensity as a function of
location on said substrate.
100. Apparatus as recited in claim 98 wherein said means for
detecting comprises a photomultiplier tube and a photon
counter.
101. Apparatus as recited in claim 99 wherein said means for
directing light further comprises a dichroic mirror, said mirror
reflecting light at a wavelength of said light source and passing
said fluoresced light.
102. Apparatus as recited in claim 100 wherein said light source is
a laser light source.
103. Apparatus as recited in claim 101 wherein said means for
storing is a programmed digital computer.
104. Apparatus as recited in claim 102 further comprising a
microscope, said light source directed at said substrate through
said microscope, said means for detecting receiving light from said
microscope.
Description
COPYRIGHT NOTICE
[0001] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
[0002] The present inventions relate to the synthesis and placement
materials at known locations. In particular, one embodiment of the
inventions provides a method and associated apparatus for preparing
diverse chemical sequences at known locations on a single substrate
surface. The inventions may be applied, for example, in the field
of preparation of oligomer, peptide, nucleic acid, oligosaccharide,
phospholipid, polymer, or drug congener preparation, especially to
create sources of chemical diversity for use in screening for
biological activity.
[0003] The relationship between structure and activity of molecules
is a fundamental issue in the study of bio-logical system.
Structure-activity relationships are important in understanding,
for example, the function of enzymes, the ways in which cells
communicate with each other, as well as cellular control and
feedback systems.
[0004] Certain macromolecules are known to interact and bind to
other molecules having a very specific three-dimensional spatial
and electronic distribution. Any large molecule having such
specificity can be considered a receptor, whether it is an enzyme
catalyzing hydrolysis of a metabolic intermediate, a cell-surface
protein mediating membrane transport of ions, a glycoprotein
serving to identify a particular cell to its neighbors, an
IgG-class antibody circulating in the plasma, an oligonucleotide
sequence of DNA in the nucleus, or the like. The various molecules
which receptors selectively bind are known as ligands.
[0005] Many assays are available for measuring the binding affinity
of known receptors and ligands, but the information which can be
gained from such experiments is often limited by the number and
type of ligands which are available. Novel ligands are sometimes
discovered by chance or by application of new techniques for the
elucidation of molecular structure, including x-ray
crystallographic analysis and recombinant genetic techniques for
proteins.
[0006] Small peptides are an exemplary system for exploring the
relationship between structure and function in biology. A peptide
is a sequence of amino acids. When the twenty naturally occurring
amino acids are condensed into polymeric molecules they form a wide
variety of three-dimensional configurations, each resulting from a
particular amino acid sequence and solvent condition. The number of
possible pentapeptides of the 20 naturally occurring amino acids,
for example, is 20.sup.5 or 3.2 million different peptides. The
likelihood that molecules of this size might be useful in
receptor-binding studies is supported by epitope analysis studies
showing that some antibodies recognize sequences as short as a few
amino acids with high specificity. Furthermore, the average
molecular weight of amino acids puts small peptides in the size
range of many currently useful pharmaceutical products.
[0007] Pharmaceutical drug discovery is one type of research which
relies on such a study of structure-activity relationships. In most
cases, contemporary pharmaceutical research can be described as the
process of discovering novel ligands with desirable patterns of
specificity for biologically important receptors. Another example
is research to discover new compounds for use in agriculture, such
as pesticides and herbicides.
[0008] Sometimes, the solution to a rational process of designing
ligands is difficult or unyielding. Prior methods of preparing
large numbers of different polymers have been painstakingly slow
when used at a scale sufficient to permit effective rational or
random screening. For example, the "Merrifield" method (J. Am.
Chem. Soc. (1963) 85: 2149-2154, which is incorporated herein by
reference for all purposes) has been used to synthesize peptides on
a solid support. In the Merrifield method, an amino acid is
covalently bonded to a support made of an insoluble polymer.
Another amino acid with an alpha protected group is reacted with
the covalently bonded amino acid to form a dipeptide. After
washing, the protective group is removed and a third amino acid
with an alpha protective group is added to the dipeptide. This
process is continued until a peptide of a desired length and
sequence is obtained. Using the Merrifield method, it is not
economically practical to synthesize more than a handful of peptide
sequences in a day.
[0009] To synthesize larger numbers of polymer sequences, it has
also been proposed to use a series of reaction vessels for polymer
synthesis. For example, a tubular reactor system may be used to
synthesize a linear polymer on a solid phase support by automated
sequential addition of reagents. This method still does not enable
the synthesis of a sufficiently large number of polymer sequences
for effective economical screening.
[0010] Methods of preparing a plurality of polymer sequences are
also known in which a foraminous container encloses a known
quantity of reactive particles, the particles being larger in size
than foramina of the container. The containers may be selectively
reacted with desired materials to synthesize desired sequences of
product molecules. As with other methods known in the art, this
method cannot practically be used to synthesize a sufficient
variety of polypeptides for effective screening.
[0011] Other techniques have also been described. These methods
include the synthesis of peptides on 96 plastic pins which fit the
format of standard microtiter plates. Unfortunately, while these
techniques have been somewhat useful, substantial problems remain.
For example, these methods continue to be limited in the diversity
of sequences which can be economically synthesized and
screened.
[0012] From the above, it is seen that an improved method and
apparatus for synthesizing a variety of chemical sequences at known
locations is desired.
SUMMARY OF THE INVENTION
[0013] An improved method and apparatus for the preparation of a
variety of polymers is disclosed.
[0014] In one preferred embodiment, linker molecules are provided
on a substrate. A terminal end of the linker molecules is provided
with a reactive functional group protected with a photoremovable
protective group. Using lithographic methods, the photoremovable
protective-group is exposed to light and removed from the linker
molecules in first selected regions. The substrate is then washed
or otherwise contacted with a first monomer that reacts with
exposed functional groups on the linker molecules. In a preferred
embodiment, the monomer is an amino acid containing a
photoremovable protective group at its amino or carboxy terminus
and the linker molecule terminates in an amino or carboxy acid
group bearing a photoremovable protective group.
[0015] A second set of selected regions is, thereafter, exposed to
light and the photoremovable protective group on the linker
molecule/protected amino acid is removed at the second set of
regions. The substrate is then contacted with a second monomer
containing a photoremovable protective group for reaction with
exposed functional groups. This process is repeated to selectively
apply monomers until polymers of a desired length and desired
chemical sequence are obtained. Photolabile groups are then
optionally removed and the sequence is, thereafter, optionally
capped. Side chain protective groups, if present, are also
removed.
[0016] By using the lithographic techniques disclosed herein, it is
possible to direct light to relatively small and precisely known
locations on the substrate. It is, therefore, possible to
synthesize polymers of a known chemical sequence at known locations
on the substrate.
[0017] The resulting substrate will have a variety of uses
including, for example, screening large numbers of polymers for
biological activity. To screen for biological activity, the
substrate is exposed to one or more receptors such as antibody
whole cells, receptors on vesicles, lipids, or any one of a variety
of other receptors. The receptors are preferably labeled with, for
example, a fluorescent marker, radioactive marker, or a labeled
antibody reactive with the receptor. The location of the marker on
the substrate is detected with, for example, photon detection or
autoradiographic techniques. Through knowledge of the sequence of
the material at the location where binding is detected, it is
possible to quickly determine which sequence binds with the
receptor and, therefore, the technique can be used to screen large
numbers of peptides. Other possible applications of the inventions
herein include diagnostics in which various antibodies for
particular receptors would be placed on a substrate and, for
example, blood sera would be screened for immune deficiencies.
Still further applications include, for example, selective "doping"
of organic materials in semiconductor devices, and the like.
[0018] In connection with one aspect of the invention an improved
reactor system for synthesizing polymers is also disclosed. The
reactor system includes a substrate mount which engages a substrate
around a periphery thereof. The substrate mount provides for a
reactor space between the substrate and the mount through or into
which reaction fluids are pumped or flowed. A mask is placed on or
focused on the substrate and illuminated so as to deprotect
selected regions of the substrate in the reactor space. A monomer
is pumped through the reactor space or otherwise contacted with the
substrate and reacts with the deprotected regions. By selectively
deprotecting regions on the substrate and flowing predetermined
monomers through the reactor space, desired polymers at known
locations may be synthesized.
[0019] Improved detection apparatus and methods are also disclosed.
The detection method and apparatus utilize a substrate having a
large variety of polymer sequences at known locations on a surface
thereof. The substrate is exposed to a fluorescently labeled
receptor which binds to one or more of the polymer sequences. The
substrate is placed in a microscope detection apparatus for
identification of locations where binding takes place. The
microscope detection apparatus includes a monochromatic or
polychromatic light source for directing light at the substrate,
means for detecting fluoresced light from the substrate, and means
for determining a location of the fluoresced light. The means for
detecting light fluoresced on the substrate may in some embodiments
include a photon counter. The means for determining a location of
the fluoresced light may include an x/y translation table for, the
substrate. Translation of the slide and data collection are
recorded and managed by an appropriately programmed digital
computer.
[0020] A further understanding of the nature and advantages of the
inventions herein may be realized by reference to the remaining
portions of the specification and the attached drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 illustrates masking and irradiation of a substrate at
a first location. The substrate is shown in cross-section;
[0022] FIG. 2 illustrates the substrate after application of a
monomer "A";
[0023] FIG. 3 illustrates irradiation of the substrate at a second
location;
[0024] FIG. 4 illustrates the substrate after application of
monomer "B";
[0025] FIG. 5 illustrates irradiation of the "A" monomer;
[0026] FIG. 6 illustrates the substrate after a second application
of "B";
[0027] FIG. 7 illustrates a completed substrate;
[0028] FIGS. 8A and 8B illustrate alternative embodiments of a
reactor system for forming a plurality of polymers on a
substrate;
[0029] FIG. 9 illustrates a detection apparatus for locating
fluorescent markers on the substrate;
[0030] FIGS. 10A-10M illustrate the method as it is applied to the
production of the trimers of monomers "A" and "B";
[0031] FIGS. 11A and 11B are fluorescence traces for standard
fluorescent beads;
[0032] FIGS. 12A and 12B are fluorescence curves for NVOC slides
not exposed and exposed to light respectively;
[0033] FIGS. 13A to 13D are fluorescence plots of slides exposed
through 100 .mu.m, 50 .mu.m, 20 .mu.m, and 10 .mu.m masks;
[0034] FIG. 14 illustrates fluorescence of a slide with the peptide
YGGFL on selected regions of its surface which has been exposed to
labeled Herz antibody specific for this sequence;
[0035] FIGS. 15A to 15D illustrate formation of and a fluorescence
plot of a slide with a checkerboard pattern of YGGFL and GGFL
exposed to labeled Herz antibody. FIG. 15C illustrates a
500.times.500 .mu.m mask which has been focused on the substrate
according to FIG. 8A while FIG. 15D illustrates a 50.times.50 .mu.m
mask placed in direct contact with the substrate in accord with
FIG. 8B;
[0036] FIG. 16 is a fluorescence plot of YGGFL and PGGFL
synthesized in a 50 .mu.m checkerboard pattern;
[0037] FIG. 17 is a fluorescence plot of YPGGFL and YGGFL
synthesized in a 50 .mu.m checkerboard pattern;
[0038] FIGS. 18A and 18B illustrate the mapping of sixteen
sequences synthesized on two different glass slides;
[0039] FIG. 19 is a fluorescence plot of the slide illustrated in
FIG. 18A; and
[0040] FIG. 20 is a fluorescence plot of the slide illustrated in
FIG. 10B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Contents
[0041] I. Glossary
[0042] II. General
[0043] III. Polymer Synthesis
[0044] IV. Details of One Embodiment of a Reactor System
[0045] V. Details of One Embodiment of a Fluorescent Detection
Device
[0046] VI. Determination of Relative Binding Strength of
Receptors
[0047] VII. Examples
[0048] A. Slide Preparation
[0049] B. Synthesis of Eight Trimers of "A" and "B"
[0050] C. Synthesis of a Dimer of an Aminopropyl Group and a
Fluorescent Group
[0051] D. Demonstration of Signal Capability
[0052] E. Determination of the Number of Molecules Per Unit
Area
[0053] F. Removal of NVOC and Attachment of a Fluorescent
Marker
[0054] G. Use of a Mask in Removal of NVOC
[0055] H. Attachment of YGGFL and Subsequent Exposure to Herz
Antibody and Goat Antimouse
[0056] I. Monomer-by-Monomer Formation of YGGFL and Subsequent
Exposure to Labeled Antibody
[0057] J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL
[0058] K. Monomer-by Monomer Synthesis of YGGFL and YPGGFL
[0059] L. Synthesis of an Array of Sixteen Different Amino Acid
Sequences and Estimation of Relative Binding Affinity to Herz
Antibody
[0060] VIII. Illustrative Alternative Embodiment
[0061] IX. Conclusion
[0062] I. Glossary
[0063] The following terms are intended to have the following
general meanings as they are used herein:
[0064] 1. Complementary: Refers to the topological compatibility or
matching together of interacting surfaces of a ligand molecule and
its receptor. Thus, the receptor and its ligand can be described as
complementary, and furthermore, the contact surface characteristics
are complementary to each other.
[0065] 2. Epitope: The portion of an antigen molecule which is
delineated by the area of interaction with the subclass of
receptors known as antibodies.
[0066] 3. Ligand: A ligand is a molecule that is recognized by a
particular receptor. Examples of ligands that can be investigated
by this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs,
lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,
proteins, and monoclonal antibodies.
[0067] 4. Monomer: A member of the set of small molecules which can
be joined together to form a polymer. The set of monomers includes
but is not restricted to, for example, the set of common L-amino
acids, the set of D-amino acids, the set of synthetic amino acids,
the set of nucleotides and the set of pentoses and hexoses. As used
herein, monomers refers to any member of a basis set for synthesis
of a polymer. For example, dimers of L-amino acids form a basis set
of 400 monomers for synthesis of polypeptides. Different basis sets
of monomers may be used at successive steps in the synthesis of a
polymer.
[0068] 5. Peptide: A polymer in which the monomers are alpha amino
acids and which are joined together through amide bonds and
alternatively referred to as a polypeptide. In the context of this
specification it should be appreciated that the amino acids may be
the L-optical isomer or the D-optical isomer. Peptides are more
than two amino acid monomers long, and often more than 20 amino
acid monomers long. Standard abbreviations for amino acids are used
(e.g., P for proline). These abbreviations are included in Stryer,
Biochemstry, Third Ed., 1988, which is incorporated herein by
reference for all purposes.
[0069] 6. Radiation: Energy which may be selectively applied
including energy having a wavelength of between 10.sup.-14 and
10.sup.4 meters including, for example, electron beam radiation,
gamma radiation, x-ray radiation, ultra-violet radiation, visible
light, infrared radiation, microwave radiation, and radio waves.
"Irradiation" refers to the application of radiation to a
surface.
[0070] 7. Receptor: A molecule that has an affinity for a given
ligand. Receptors may be naturally-occuring or manmade molecules.
Also, they can be employed in their unaltered state or as
aggregates with other species. Receptors may be attached,
covalently or noncovalently, to a binding member, either directly
or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted
to, antibodies, cell membrane receptors, monoclonal antibodies and
antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic
acids, peptides, cofactors, lectins, sugars, polysaccharides,
cells, cellular membranes, and organelles. Receptors are sometimes
referred to in the art as anti-ligands. As the term receptors is
used herein, no difference in meaning is intended. A "Ligand
Receptor Pair" is formed when two macromolecules have combined
through molecular recognition to form a complex.
[0071] Other examples of receptors which can be investigated by
this invention include but are not restricted to:
[0072] a) Microorganism receptors: Determination of ligands which
bind to receptors, such as specific transport proteins or enzymes
essential to survival of microorganisms, is useful in a new class
of antibiotics. Of particular value would be antibiotics against
opportunistic fungi, protozoa, and those bacteria resistant to the
antibiotics in current use.
[0073] b) Enzymes: For instance, the binding site of enzymes such
as the enzymes responsible for cleaving neurotransmitters;
determination of ligands which bind to certain receptors to
modulate the action of the enzymes which cleave the different
neurotransmitters is useful in the development of drugs which can
be used in the treatment of disorders of neurotransmission.
[0074] c) Antibodies: For instance, the invention may be useful in
investigating the ligand-binding site on the antibody molecule
which combines with the epitope of an antigen of interest;
determining a sequence that mimics an antigenic epitope may lead to
the development of vaccines of which the immunogen is based on one
or more of such sequences or lead to the development of related
diagnostic agents or compounds useful in therapeutic treatments
such as for auto-immune diseases (e.g., by blocking the binding of
the "self" antibodies).
[0075] d) Nucleic Acids: Sequences of nucleic acids may be
synthesized to establish DNA or RNA binding sequences.
[0076] e) Catalytic Polypeptides: Polymers, preferably
polypeptides, which are capable of promoting a chemical reaction
involving the conversion of one or more reactants to one or more
products. Such polypeptides generally include a binding site
specific for at least one reactant or reaction intermediate and an
active functionality proximate to the binding site, which
functionality is capable of chemically modifying the bound
reactant. Catalytic polypeptides are described in, for example,
U.S. application Ser. No. 404,920, which is incorporated herein by
reference for all purposes.
[0077] f) Hormone receptors: For instance, the receptors for
insulin and growth hormone. Determination of the ligands which bind
with high affinity to a receptor is useful in the development of,
for example, an oral replacement of the daily injections which
diabetics must take to relieve the symptoms of diabetes, and in the
other case, a replacement for the scarce human growth hormone which
can only be obtained from cadavers or by recombinant DNA
technology. Other examples are the vasoconstrictive hormone
receptors; determination of those ligands which bind to a receptor
may lead to the development of drugs to control blood pressure.
[0078] g) Opiate receptors: Determination of ligands which bind to
the opiate receptors in the brain is useful in the development of
less-addictive replacements for morphine and related drugs.
[0079] 8. Substrate: A material having a rigid or semi-rigid
surface. In many embodiments, at least one surface of the substrate
will be substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
polymers with, for example, wells, raised regions, etched trenches,
or the like. According to other embodiments, small beads may be
provided on the surface which may be released upon completion of
the synthesis.
[0080] 9. Protective Group: A material which is bound to a monomer
unit and which may be spatially removed upon selective exposure to
an activator such as electromagnetic radiation. Examples of
protective groups with utility herein include Nitroveratryloxy
carbonyl, Nitrobenzyloxy carbonyl, Dimethyl dimethoxybenzyloxy
carbonyl, 5-Bromo-7-nitroindolinyl, o-Hydroxy-.alpha.-methyl
cinnamoyl, and 2-oxymethylene anthraquinone. Other examples of
activators include ion beams, electric fields magnetic fields,
electron beams, x-ray, and the like.
[0081] 10. Predefined Region: A predefined region is a localized
area on a surface which is, was, or is intended to be activated for
formation of a polymer. The predefined region may have any
convenient shape, e.g., circular, rectangular, elliptical,
wedge-shaped, etc. For the sake of brevity herein, "predefined
regions" are sometimes referred to simply as "regions."
[0082] 11. Substantially Pure: A polymer is considered to be
"substantially pure" within a predefined region of a substrate when
it exhibits characteristics that distinguish it from other
predefined regions. Typically, purity will be measured in terms of
biological activity or function as a result of uniform sequence.
Such characteristics will typically be measured by way of binding
with a selected ligand or receptor.
[0083] II. General
[0084] The present invention provides methods and apparatus for the
preparation and use of a substrate having a plurality of polymer
sequences in predefined regions. The invention is described herein
primarily with regard to the preparation of molecules containing
sequences of amino acids, but could readily be applied in the
preparation of other polymers. Such polymers include, for example,
both linear and cyclic polymers of nucleic acids, polysaccharides,
phospholipids, and peptides having either .alpha.-, .beta.-, or
.omega.-amino acids, hetero-polymers in which a known drug is
covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides polysiloxanes, polyimides, polyacetates, or
other polymers which will be apparent upon review of this
disclosure. In a preferred embodiment, the invention herein is used
in the synthesis of peptides.
[0085] The prepared substrate may, for example, be used in
screening a variety of polymers as ligands for binding with a
receptor, although it will be apparent that the invention could be
used for the synthesis of a receptor for binding with a ligand. The
substrate disclosed herein will have a wide variety of other uses.
Merely by way of example, the invention herein can be used in
determining peptide and nucleic acid sequences which bind to
proteins, finding sequence-specific binding drugs, identifying
epitopes recognized by antibodies, and evaluation of a variety of
drugs for clinical and diagnostic applications, as well as
combinations of the above.
[0086] The invention preferably provides for the use of a substrate
"S" with a surface. Linker molecules "L" are optionally provided on
a surface of the substrate. The purpose of the linker molecules, in
some embodiments, is to facilitate receptor recognition of the
synthesized polymers.
[0087] Optionally, the linker molecules may be chemically protected
for storage purposes. A chemical storage protective group such as
t-BOC (t-butoxycarbonyl) may be used in some embodiments. Such
chemical protective groups would be chemically removed upon
exposure to, for example, acidic solution and would serve to
protect the surface during storage and be removed prior to polymer
preparation.
[0088] On the substrate or a distal end of the linker molecules, a
functional group with a protective group P.sub.0 is provided. The
protective group P.sub.0 may be removed upon exposure to radiation,
electric fields, electric currents, or other activators to expose
the functional group.
[0089] In a preferred embodiment, the radiation is ultraviolet
(UV), infrared (IR), or visible light. As more fully described
below, the protective group may alternatively be an
electrochemically-sensitive group which may be removed in the
presence of an electric field. In still further alternative
embodiments, ion beams, electron beams, or the like may be used for
deprotection.
[0090] In some embodiments, the exposed regions and, therefore, the
area upon which each distinct polymer sequence is synthesized are
smaller than about 1 cm.sup.2 or less than 1 mm.sup.2. In preferred
embodiments the exposed area is less than about 10,000 .mu.m.sup.2
or, more preferably, less than 100 .mu.m.sup.2 and may, in some
embodiments, encompass the binding site for as few as a single
molecule. Within these regions, each polymer is preferably
synthesized in a substantially pure form.
[0091] Concurrently or after exposure of a known region of the
substrate to light, the surface is contacted with a first monomer
unit M.sub.1 which reacts with the functional group which has been
exposed by the deprotection step. The first monomer includes a
protective group P.sub.1. P.sub.1 may or may not be the same as
P.sub.0.
[0092] Accordingly, after a first cycle, known first regions of the
surface may comprise the sequence:
[0093] S-L-M.sub.1-P.sub.1
[0094] while remaining regions of the surface comprise the
sequence:
[0095] S-L-P.sub.0.
[0096] Thereafter, second regions of the surface (which may include
the first region) are exposed to light and contacted with a second
monomer M.sub.2 (which may or may not be the same as M.sub.1)
having a protective group P.sub.2. P.sub.2 may or may not be the
same as P.sub.0 and P.sub.1. After this second cycle, different
regions of the substrate may comprise one or more of the following
sequences:
[0097] S-L-M.sub.1-M.sub.2-P.sub.2
[0098] S-L-M.sub.2-P.sub.2
[0099] S-L-M.sub.1-P.sub.1 and/or
[0100] S-L-P.sub.0.
[0101] The above process is repeated until the substrate includes
desired polymers of desired lengths. By controlling the locations
of the substrate exposed to light and the reagents exposed to the
substrate following exposure, the location of each sequence will be
known.
[0102] Thereafter, the protective groups are removed from some or
all of the substrate and the sequences are, optionally, capped with
a capping unit C. The process results in a substrate having a
surface with a plurality of polymers of the following general
formula:
S-[L]-(M.sub.i)-(M.sub.j)-(M.sub.k) . . . (M.sub.x)-[C]
[0103] where square brackets indicate optional groups, and M.sub.i
. . . M.sub.x indicates any sequence of monomers. The number of
monomers could cover a wide variety of values, but in a preferred
embodiment they will range from 2 to 100.
[0104] In some embodiments a plurality of locations on the
substrate polymers are to contain a common monomer subsequence. For
example, it may be desired to synthesize a sequence
S-M.sub.1-M.sub.2-M.sub.3 at first locations and a sequence
S-M.sub.4-M.sub.2-M.sub.3 at second locations. The process would
commence with irradiation of the first locations followed by
contacting with M.sub.1-P, resulting in the sequence S-M.sub.1-P at
the first location. The second locations would then be irradiated
and contacted with M.sub.4-P, resulting in the sequence S-M.sub.4-P
at the second locations. Thereafter both the first and second
locations would be irradiated and contacted with the dimer
M.sub.2-M.sub.3, resulting in the sequence
S-M.sub.1-M.sub.2-M.sub.3 at the first locations and
S-M.sub.4-M.sub.2-M.sub.3 at the second locations. Of course,
common subsequences of any length could be utilized including those
in a range of 2 or more monomers, 2 to 100 monomers, 2 to 20
monomers, and a most preferred range of 2 to 3 monomers.
[0105] According to other embodiments, a set of masks is used for
the first monomer layer and, thereafter, varied light wavelengths
are used for selective deprotection. For example, in the process
discussed above, first regions are first exposed through a mask and
reacted with a first monomer having a first protective group
P.sub.1, which is removable upon exposure to a first wavelength of
light (e.g., IR). Second regions are masked and reacted with a
second monomer having a second protecive group P.sub.2, which is
removable upon exposure to a second wavelength of light (e.g., UV).
Thereafter, masks become unnecessary in the synthesis because the
entire substrate may be exposed alternatively to the first and
second wavelengths of light in the deprotection cycle.
[0106] The polymers prepared on a substrate according to the above
methods will have a variety of uses including, for example,
screening for biological activity. In such screening activities,
the substrate containing the sequences is exposed to an unlabeled
or labeled receptor such as an antibody, receptor on a cell,
phospholipid vesicle, or any one of a variety of other receptors.
In one preferred embodiment the polymers are exposed to a first,
unlabeled receptor of interest and, thereafter, exposed to a
labeled receptor-specific recognition element, which is, for
example, an antibody. This process will provide signal
amplification in the detection stage.
[0107] The receptor molecules may bind with one or more polymers on
the substrate. The presence of the labeled receptor and, therefore,
the presence of a sequence which binds with the receptor is
detected in a preferred embodiment through the use of
autoradiography, detection of fluorescence with a charge-coupled
device, fluorescence microscopy, or the like. The sequence of the
polymer at the locations where the receptor binding is detected may
be used to determine all or part of a sequence which is
complementary to the receptor.
[0108] Use of the invention herein is illustrated primarily with
reference to screening for biological activity. The invention will,
however, find many other uses. For example, the invention may be
used in information storage (e.g., on optical disks), production of
molecular electronic devices, production of stationary phases in
separation sciences, production of dyes and brightening agents,
photography, and in immobilization of cells, proteins, lectins,
nucleic acids, polysaccharides and the like in patterns on a
surface via molecular recognition of specific polymer sequences. By
synthesizing the same compound in adjacent, progressively differing
concentrations, a gradient will be established to control
chemotaxis or to develop diagnostic dipsticks which, for example,
titrate an antibody against an increasing amount of antigen. By
synthesizing several catalyst molecules in close proximity, more
efficient multistep conversions may be achieved by "coordinate
immobilization." Coordinate immobilization also may be used for
electron transfer systems, as well as to provide both structural
integrity and other desirable properties to materials such as
lubrication, wetting, etc.
[0109] According to alternative embodiments, molecular
biodistribution or pharmacokinetic properties may be examined. For
example, to assess resistance to intestinal or serum proteases,
polymers may be capped with a fluorescent tag and exposed to
biological fluids of interest.
[0110] III. Polymer Synthesis
[0111] FIG. 1 illustrates one embodiment of the invention disclosed
herein in which a substrate 2 is shown in cross-section.
Essentially, any conceivable substrate may be employed in the
invention. The substrate may be biological, nonbiological, organic,
inorganic, or a combination of any of these, existing as particles,
strands, precipitates, gels, sheets, tubing, spheres, containers,
capillaries, pads, slices, films, plates, slides, etc. The
substrate may have any convenient shape, such as a disc, square,
sphere, circle, etc. The substrate is preferably flat but may take
on a variety of alternative surface configurations. For example,
the substrate may contain raised or depressed regions on which the
synthesis takes place. The substrate and its surface preferably
form a rigid support on which to carry out the reactions described
herein. The substrate and its surface is also chosen to provide
appropriate light-absorbing characteristics. For instance, the
substrate may be a polymerized Langmuir Blodgett film,
functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2, SiN.sub.4,
modified silicon, or any one of a wide variety of gels or polymers
such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,
polystyrene, polycarbonate, or combinations thereof. Other
substrate materials will be readily apparent to those of skill in
the art upon review of this disclosure. In a preferred embodiment
the substrate is flat glass or single-crystal silicon with surface
relief features of less than 10 .ANG..
[0112] According to some embodiments, the surface of the substrate
is etched using well known techniques to provide for desired
surface features. For example, by way of the formation of trenches,
v-grooves, mesa structures, or the like, the synthesis regions may
be more closely placed within the focus point of impinging light,
be provided with reflective "mirror" structures for maximization of
light collection from fluorescent sources, or the like.
[0113] Surfaces on the solid substrate will usually, though not
always, be composed of the same material as the substrate. Thus,
the surface may be composed of any of a wide variety of materials,
for example, polymers, plastics, resins, polysaccharides, silica or
silica-based materials, carbon, metals, inorganic glasses,
membranes, or any of the above-listed substrate materials. In some
embodiments the surface may provide for the use of caged binding
members which are attached firmly to the surface of the substrate
in accord with the teaching of copending application Ser. No.
404,920, previously incorporated herein by reference. Preferably,
the surface will contain reactive groups, which could be carboxyl,
amino, hydroxyl, or the like. Most preferably, the surface will be
optically transparent and will have surface Si--OH functionalities,
such as are found on silica surfaces.
[0114] The surface 4 of the substrate is preferably provided with a
layer of linker molecules 6, although it will be understood that
the linker molecules are not required elements of the invention.
The linker molecules are preferably of sufficient length to permit
polymers in a completed substrate to interact freely with molecules
exposed to the substrate. The linker molecules should be 6-50 atoms
long to provide sufficient exposure. The linker molecules may be,
for example, aryl acetylene, ethylene glycol oligomers containing
2-10 monomer units, diamines, diacids, amino acids, or combinations
thereof. Other linker molecules may be used in light of this
disclsoure.
[0115] According to alternative embodiments, the linker molecules
are selected based upon their hydrophilic/hydrophobic properties to
improve presentation of synthesized polymers to certain receptors.
For example, in the case of a hydrophilic receptor, hydrophilic
linker molecules will be preferred so as to permit the receptor to
more closely approach the synthesized polymer.
[0116] According to another alternative embodiment, linker
molecules are also provided with a photocleavable group at an
intermediate position. The photocleavable group is preferably
cleavable at a wavelength different from the protective group. This
enables removal of the various polymers following completion of the
synthesis by way of exposure to the different wavelengths of
light.
[0117] The linker-molecules can be attached to the substrate via
carbon-carbon bonds using, for example,
(poly)trifluorochloroethylene surfaces, or preferably, by siloxane
bonds (using, for example, glass or silicon oxide surfaces).
Siloxane bonds with the surface of the substrate may be formed in
one embodiment via reactions of linker molecules bearing
trichlorosilyl groups. The linker molecules may optionally be
attached in an ordered array, i.e., as parts of the head groups in
a polymerized Langmuir Blodgett film. In alternative embodiments,
the linker molecules are adsorbed to the surface of the
substrate.
[0118] The linker molecules and monomers used herein are provided
with a functional group to which is bound a protective group.
Preferably, the protective group is on the distal or terminal end
of the linker molecule opposite the substrate. The protective group
may be either a negative protective group (i.e., the protective
group renders the linker molecules less reactive with a monomer
upon exposure) or a positive protective group (i.e., the protective
group renders the linker molecules more reactive with a monomer
upon exposure). In the case of negative protective groups an
additional step of reactivation will be required. In some
embodiments, this will be done by heating.
[0119] The protective group on the linker-molecules may be selected
from a wide variety of positive light-reactive groups preferably
including nitro aromatic compounds such as o-nitrobenzyl
derivatives or benzylsulfonyl. In a preferred embodiment,
6-nitroveratryloxycarbonyl (NVOC), 2-nitrobenzyloxycarbonyl (NBOC)
or .alpha.,.alpha.-dimethyl-dimethoxybenz- yloxycarbonyl (DDZ) is
used. In one embodiment, a nitro aromatic compound containing a
benzylic hydrogen ortho to the nitro group is used, i.e., a
chemical of the form: 1
[0120] where R.sub.1 is alkoxy, alkyl, halo, aryl, alkenyl, or
hydrogen; R.sub.2 is alkoxy, alkyl, halo, aryl, nitro, or hydrogen;
R.sub.3 is alkoxy, alkyl, halo, nitro, aryl, or hydrogen; R.sub.4
is alkoxy, alkyl, hydrogen, aryl, halo, or nitro; and R.sub.5 is
alkyl, alkynyl, cyano, alkoxy, hydrogen, halo, aryl, or alkenyl.
Other materials which may be used include o-hydroxy-.alpha.-methyl
cinnamoyl derivatives. Photoremovable protective groups are
described in, for example, Patchornik, J. Am. Chem. Soc. (1970) 92:
6333 and Amit et al., J. Org. Chem. (1974) 39: 192, both of which
are incorporated herein by reference.
[0121] In an alternative embodiment the positive reactive group is
activated for reaction with reagents in solution. For example, a
5-bromo-7-nitro indoline group, when bound to a carbonyl, undergoes
reaction upon exposure to light at 420 nm.
[0122] In a second alternative embodiment, the reactive group on
the linker molecule is selected from a wide variety of negative
light-reactive groups including a cinammate group.
[0123] Alternatively, the reactive group is activated or
deactivated by electron beam lithography, x-ray lithography, or any
other radiation. Suitable reactive groups for electron beam
lithography include sulfonyl. Other methods may be used including,
for example, exposure to a current source. Other reactive groups
and methods of activation may be used in light of this
disclosure.
[0124] As shown in FIG. 1, the linking molecules are preferably
exposed to, for example, light through a suitable mask 8 using
photolithographic techniques of the type known in the semiconductor
industry and described in, for example, Sze, VLSI Technology,
McGraw-Hill (1983), and Mead et al., Introduction to VLSI Systems,
Addison-Wesley (1980), which are incorporated herein by reference
for all purposes. The light may be directed at either the surface
containing the protective groups or at the back of the substrate,
so long as the substrate is transparent to the wavelength of light
needed for removal of the protective groups. In the embodiment
shown in FIG. 1, light is directed at the surface of the substrate
containing the protective groups. FIG. 1 illustrates the use of
such masking techniques as they are applied to a positive reactive
group so as to activate linking molecules and expose functional
groups in areas 10a and 10b.
[0125] The mask 8 is in one embodiment a transparent support
material selectively coated with a layer of opaque material.
Portions of the opaque material are removed, leaving opaque
material in the precise pattern desired on the substrate surface.
The mask is brought into close proximity with, imaged on, or
brought directly into contact with the substrate surface as shown
in FIG. 1. "Openings" in the mask correspond to locations on the
substrate where it is desired to remove photoremovable protective
groups from the substrate. Alignment may be performed using
conventional alignment techniques in which alignment marks (not
shown) are used to accurately overlay successive masks with
previous patterning steps, or more sophisticated techniques may be
used. For example, interferometric techniques such as the one
described in Flanders et al., "A New Interferometric Alignment
Technique," App. Phys. Lett. (1977) 31: 426-428, which is
incorporated herein by reference, may be used.
[0126] To enhance contrast of light applied to the substrate, it is
desirable to provide contrast enhancement materials between the
mask and the substrate according to some embodiments. This contrast
enhancement layer may comprise a molecule which is decomposed by
light such as quinone diazid or a material which is transiently
bleached at the wavelength of interest. Transient bleaching of
materials will allow greater penetration where light is applied,
thereby enhancing contrast. Alternatively, contrast enhancement may
be provided by way of a cladded fiber optic bundle.
[0127] The light may be from a conventional incandescent source, a
laser, a laser diode, or the like. If non-collimated sources of
light are used it may be desirable to provide a thick- or
multi-layered mask to prevent spreading of the light onto the
substrate. It may, further, be desirable in some embodiments to
utilize groups which are sensitive to different wavelengths to
control synthesis. For example, by using groups which are sensitive
to different wavelengths, it is possible to select branch positions
in the synthesis of a polymer or eliminate certain masking steps.
Several reactive groups along with their corresponding wavelengths
for deprotection are provided in Table 1.
1 TABLE 1 Approximate Deprotection Group Wavelength
Nitroveratryloxy carbonyl (NVOC) UV (300-400 nm) Nitrobenzyloxy
carbonyl (NBOC) UV (300-350 nm) Dimethyl dimethoxybenzyloxy
carbonyl UV (280-300 nm) 5-Bromo-7-nitroindolinyl UV (420 nm)
o-Hydroxy-.alpha.-methyl cinnamoyl UV (300-350 nm) 2-Oxymethylene
anthraquinone UV (350 nm)
[0128] While the invention is illustrated primarily herein by way
of the use of a mask to illuminate selected regions the substrate,
other techniques may also be used. For example, the substrate may
be translated under a modulated laser or diode light source. Such
techniques are discussed in, for example, U.S. Pat. No. 4,719,615
(Feyrer et al.), which is incorporated herein by reference. In
alternative embodiments a laser galvanometric scanner is utilized.
In other embodiments, the synthesis may take place on or in contact
with a conventional liquid crystal (referred to herein as a "light
valve") or fiber optic light sources. By appropriately modulating
liquid crystals, light may be selectively controlled so as to
permit light to contact selected regions of the substrate.
Alternatively, synthesis may take place on the end of a series of
optical fibers to which light is selectively applied. Other means
of controlling the location of light exposure will be apparent to
those of skill in the art.
[0129] The substrate may be irradiated either in contact or not in
contact with a solution (not shown) and is, preferably, irradiated
in contact with a solution. The solution contains reagents to
prevent the by-products formed by irradiation from interfering with
synthesis of the polymer according to some embodiments. Such
by-products might include, for example, carbon dioxide,
nitrosocarbonyl compounds, styrene derivatives, indole derivatives,
and products of their photochemical reactions. Alternatively, the
solution may contain reagents used to match the index of refraction
of the substrate. Reagents added to the solution may further
include, for example, acidic or basic buffers, thiols, substituted
hydrazines and hydroxylamines, reducing agents (e.g., NADH) or
reagents known to react with a given functional group (e.g., aryl
nitroso+glyoxylic acid.fwdarw.aryl formhydroxamate+CO.sub.2).
[0130] Either concurrently with or after the irradiation step, the
linker molecules are washed or otherwise contacted with a first
monomer, illustrated by "A" in regions 12a and 12b in FIG. 2. The
first monomer reacts with the activated functional groups of the
linkage molecules which have been exposed to light. The first
monomer, which is preferably an amino acid, is also provided with a
photoprotective group. The photoprotective group on the monomer may
be the same as or different than the protective group used in the
linkage molecules, and may be selected from any of the
above-described protective groups. In one embodiment, the
protective groups for the A monomer is selected from the group NBOC
and NVOC.
[0131] As shown in FIG. 3, the process of irradiating is thereafter
repeated, with a mask repositioned so as to remove linkage
protective groups and expose functional groups in regions 14a and
14b which are illustrated as being regions which were protected in
the previous masking step. As an alternative to repositioning of
the first mask, in many embodiments a second mask will be utilized.
In other alternative embodiments, some steps may provide for
illuminating a common region in successive steps. As shown in FIG.
3, it may be desirable to provide separation between irradiated
regions. For example, separation of about 1-5 .mu.m may be
appropriate to account for alignment tolerances.
[0132] As shown in FIG. 4, the substrate is then exposed to a
second protected monomer "B," producing B regions 16a and 16b.
Thereafter, the substrate is again masked so as to remove the
protective groups and expose reactive groups on A region 12a and B
region 16b. The substrate is again exposed to monomer B, resulting
in the formation of the structure shown in FIG. 6. The dimers B-A
and B-B have been produced on the substrate.
[0133] A subsequent series of masking and contacting steps similar
to those described above with A (not shown) provides the structure
shown in FIG. 7. The process provides all possible dimers of B and
A, i.e., B-A, A-B, A-A, and B-B.
[0134] The substrate, the area of synthesis, and the area for
synthesis of each individual polymer could be of any size or shape.
For example, squares, ellipsoids, rectangles, triangles, circles,
or portions thereof, along with irregular geometric shapes, may be
utilized. Duplicate synthesis areas may also be applied to a single
substrate for purposes of redundancy.
[0135] In one embodiment the regions 12 and 16 on the substrate
will have a surface area of between about 1 cm.sup.2 and 10.sup.-10
cm.sup.2. In some embodiments the regions 12 and 16 have areas of
less than about 10.sup.-1 cm.sup.2, 10.sup.-2 cm.sup.2, 10.sup.-3
cm.sup.2, 10.sup.-4 cm.sup.2, 10.sup.-5 cm.sup.3, 10.sup.-6
cm.sup.2, 10.sup.-7 cm.sup.2, 10.sup.-8 cm.sup.2, or 10.sup.-10
cm.sup.2. In a preferred embodiment, the regions 12 and 16 are
between about 10.times.10 .mu.m and 500.times.500 .mu.m.
[0136] In some embodiments a single substrate supports more than
about 10 different monomer sequences and perferably more than about
100 different monomer sequences, although in some embodiments more
than about 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, or
10.sup.8 different sequences are provided on a substrate. Of
course, within a region of the substrate in which a monomer
sequence is synthesized, it is preferred that the monomer sequence
be substantially pure. In some embodiments, regions of the
substrate contain polymer sequences which are at least about 1%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,
90%, 95%, 96%, 97%, 98%, or 99% pure.
[0137] According to some embodiments, several sequences are
intentionally provided within a single region so as to provide an
initial screening for biological activity, after which materials
within regions exhibiting significant binding are further
evaluated.
[0138] IV. Details of One Embodiment of a Reactor System
[0139] FIG. 8A schematically illustrates a preferred embodiment of
a reactor system 100 for synthesizing polymers on the prepared
substrate in accordance with one aspect of the invention. The
reactor system includes a body 102 with a cavity 104 on a surface
thereof. In preferred embodiments the cavity 104 is between about
50 and 1000 .mu.m deep with a depth of about 500 .mu.m
preferred.
[0140] The bottom of the cavity is preferably provided with an
array of ridges 106 which extend both into the plane of the Figure
and parallel to the plane of the Figure. The ridges are preferably
about 50 to 200 .mu.m deep and spaced at about 2 to 3 mm. The
purpose of the ridges is to generate turbulent flow for better
mixing. The bottom surface of the cavity is preferably light
absorbing so as to prevent reflection of impinging light.
[0141] A substrate 112 is mounted above the cavity 104. The
substrate is provided along its bottom surface 114 with a
photoremovable protective group such as NVOC with or without an
intervening linker molecule. The substrate is preferably
transparent to a wide spectrum of light, but in some embodiments is
transparent only at a wavelength at which the protective group may
be removed (such as UV in the case of NVOC). The substrate in some
embodiments is a conventional microscope glass slide or cover slip.
The substrate is preferably as thin as possible, while still
providing adequate physical support. Preferably, the substrate is
less than about 1 mm thick, more preferably less than 0.5 mm thick,
more preferably less than 0.1 mm thick, and most preferably less
than 0.05 mm thick. In alternative preferred embodiments, the
substrate is quartz or silicon.
[0142] The substrate and the body serve to seal the cavity except
for an inlet port 108 and an outlet port 110. The body and the
substrate may be mated for sealing in some embodiments with one or
more gaskets. According to a preferred embodiment, the body is
provided with two concentric gaskets and the intervening space is
held at vacuum to ensure mating of the substrate to the
gaskets.
[0143] Fluid is pumped through the inlet port into the cavity by
way of a pump 116 which may be, for example, a model no. B-120-S
made by Eldex Laboratories. Selected fluids are circulated into the
cavity by the pump, through the cavity, and out the outlet for
recirculation or disposal. The reactor may be subjected to
ultrasonic radiation and/or heated to aid in agitation in some
embodiments.
[0144] Above the substrate 112, a lens 120 is provided which may
be, for example, a 2" 100 mm focal length fused silica lens. For
the sake of a compact system, a reflective mirror 122 may be
provided for directing light from a light source 124 onto the
substrate. Light source 124 may be, for example, a Xe(Hg) light
source manufactured by Oriel and having model no. 66024. A second
lens 126 may be provided for the purpose of projecting a mask image
onto the substrate in combination with lens 112. This form of
lithography is referred to herein as projection printing. As will
be apparent from this disclosure, proximity printing and the like
may also be used according to some embodiments.
[0145] Light from the light source is permitted to reach only
selected locations on the substrate as a result of mask 128. Mask
128 may be, for example, a glass slide having etched chrome
thereon. The mask 128 in one embodiment is provided with a grid of
transparent locations and opaque locations. Such masks may be
manufactured by, for example, Photo Sciences, Inc. Light passes
freely through the transparent regions of the mask, but is
reflected from or absorbed by other regions. Therefore, only
selected regions of the substrate are exposed to light.
[0146] As discussed above, light valves (LCD's) may be used as an
alternative to conventional masks to selectively expose regions of
the substrate. Alternatively, fiber optic faceplates such as those
available from Schott Glass, Inc, may be used for the purpose of
contrast enhancement of the mask or as the sole means of
restricting the region to which light is applied. Such faceplates
would be placed directly above or on the substrate in the reactor
shown in FIG. 8A. In still further embodiments, flys-eye lenses,
tapered fiber optic faceplates, or the like, may be used for
contrast enhancement.
[0147] In order to provide for illumination of regions smaller than
a wavelength of light, more elaborate techniques may be utilized.
For example, according to one preferred embodiment, light is
directed at the substrate by way of molecular microcrystals on the
tip of, for example, micropipettes. Such devices are disclosed in
Lieberman et al., "A Light Source Smaller Than the Optical
Wavelength," Science (1990) 247: 59-61, which is incorporated
herein by reference for all purposes.
[0148] In operation, the substrate is placed on the cavity and
sealed thereto. All operations in the process of preparing the
substrate are carried out in a room lit primarily or entirely by
light of a wavelength outside of the light range at which the
protective group is removed. For example, in the case of NVOC, the
room should be lit with a conventional dark room light which
provides little or no UV light. All operations are preferably
conducted at about room temperature.
[0149] A first, deprotection fluid (without a monomer) is
circulated through the cavity. The solution preferably is of 5 mM
sulfuric acid in dioxane solution which serves to keep exposed
amino groups protonated and decreases their reactivity with
photolysis by-products. Absorptive materials such as
N,N-diethylamino 2,4-dinitrobenzene, for example, may be included
in the deprotection fluid which serves to absorb light and prevent
reflection and unwanted photolysis.
[0150] The slide is, thereafter, positioned in a light raypath from
the mask such that first locations on the substrate are illuminated
and, therefore, deprotected. In preferred embodiments the substrate
is illuminated for between about 1 and 15 minutes with a preferred
illumination time of about 10 minutes at 10-20 mW/cm.sup.2 with 365
nm light. The slides are neutralized (i.e., brought to a pH of
about 7) after photolysis with, for example, a solution of
di-isopropylethylamine (DIEA) in methylene chloride for about 5
minutes.
[0151] The first monomer is then placed at the first locations on
the substrate. After irradiation, the slide is removed, treated in
bulk, and then reinstalled in the flow cell. Alternatively, a fluid
containing the first monomer, preferably also protected by a
protective group, is circulated through the cavity by way of pump
116. If, for example, it is desired to attach the amino acid Y to
the substrate at the first locations, the amino acid Y (bearing a
protective group on its .alpha.-nitrogen), along with reagents used
to render the monomer reactive, and/or a carrier, is circulated
from a storage container 118, through the pump, through the cavity,
and back to the inlet of the pump.
[0152] The monomer carrier solution is, in a preferred embodiment,
formed by mixing of a first solution (referred to herein as
solution "A") and a second solution (referred to herein as solution
"B"). Table 2 provides an illustration of a mixture which may be
used for solution A.
2TABLE 2 Representative Monomer Carrier Solution "A" 100 mg NVOC
amino protected amino acid 37 mg HOBT (1-Hydroxybenzotriazole) 250
.mu.l DMF (Dimethylformamide) 86 .mu.l DIEA
(Diisopropylethylamine)
[0153] The composition of solution B is illustrated in Table 3.
Solutions A and B are mixed and allowed to react at room
temperature for about 8 minutes, then diluted with 2 ml of DMF, and
500 .mu.l are applied to the surface of the slide or the solution
is circulated through the reactor system and allowed to react for
about 2 hours at room temperature. The slide is then washed with
DMF, methylene chloride and ethanol.
3TABLE 3 Representative Monomer Carrier Solution "B" 250 .mu.l DMF
111 mg BOP (Benzotriazolyl-n-oxy-tris(dimethylamino)
phosphoniumhexafluoroph- osphate)
[0154] As the solution containing the monomer to be attached is
circulated through the cavity, the amino acid or other monomer will
react at its carboxy terminus with amino groups on the regions of
the substrate which have been deprotected. Of course, while the
invention is illustrated by way of circulation of the monomer
through the cavity, the invention could be practiced by way of
removing the slide from the reactor and submersing it in an
appropriate monomer solution.
[0155] After addition of the first monomer, the solution containing
the first amino acid is then purged from the system. After
circulation of a sufficient amount of the DMF/methylene chloride
such that removal of the amino acid can be assured (e.g., about
50.times. times the volume of the cavity and carrier lines), the
mask or substrate is repositioned, or a new mask is utilized such
that second regions on the substrate will be exposed to light and
the light 124 is engaged for a second exposure. This will deprotect
second regions on the substrate and the process is repeated until
the desired polymer sequences have been synthesized.
[0156] The entire derivatized substrate is then exposed to a
receptor of interest, preferably labeled with, for example, a
fluorescent marker, by circulation of a solution or suspension of
the receptor through the cavity or by contacting the surface of the
slide in bulk. The receptor will preferentially bind to certain
regions of the substrate which contain complementary sequences.
[0157] Antibodies are typically suspended in what is commonly
referred to as "supercocktail," which may be, for example, a
solution of about 1% BSA (bovine serum albumin), 0.5% Tween in PBS
(phosphate buffered saline) buffer. The antibodies are diluted into
the supercocktail buffer to a final concentration of, for example,
about 0.1 to 4 .mu.g/ml.
[0158] FIG. 8B illustrates an alternative preferred embodiment of
the reactor shown in FIG. 8A. According to this embodiment, the
mask 128 is placed directly in contact with the substrate.
Preferably, the etched portion of the mask is placed face down so
as to reduce the effects of light dispersion. According to this
embodiment, the imaging lenses 120 and 126 are not necessary
because the mask is brought into close proximity with the
substrate.
[0159] For purposes of increasing the signal-to-noise ratio of the
technique, some embodiments of the invention provide for exposure
of the substrate to a first labeled or unlabeled receptor followed
by exposure of a labeled, second receptor (e.g., an antibody) which
binds at multiple sites on the first receptor. If, for example, the
first receptor is an antibody derived from a first species of an
animal, the second receptor is an antibody derived from a second
species directed to epitopes associated with the first species. In
the case of a mouse antibody, for example, fluorescently labeled
goat antibody or antiserum which is antimouse may be used to bind
at multiple sites on the mouse antibody, providing several times
the fluorescence compared to the attachment of a single mouse
antibody at each binding site. This process may be repeated again
with additional antibodies (e.g., goat-mouse-goat, etc.) for
further signal amplification.
[0160] In preferred embodiments an ordered sequence of masks is
utilized. In some embodiments it is possible to use as few as a
single mask to synthesize all of the possible polymers of a given
monomer set.
[0161] If, for example, it is desired to synthesize all 16
dinucleotides from four bases, a 1 cm square synthesis region is
divided conceptually into 16 boxes, each 0.25 cm wide. Denote the
four monomer units by A, B, C, and D. The first reactions are
carried out in four vertical columns, each 0.25 cm wide. The first
mask exposes the left-most column of boxes, where A is coupled. The
second mask exposes the next column, where B is coupled; followed
by a third mask, for the C column; and a final mask that exposes
the right-most column, for D. The first, second, third, and fourth
masks may be a single mask translated to different locations.
[0162] The process is repeated in the horizontal direction for the
second unit of the dimer. This time, the masks allow exposure of
horizontal rows, again 0.25 cm wide. A, B, C, and D are
sequentially coupled using masks that expose horizontal fourths of
the reaction area. The resulting substrate contains all 16
dinucleotides of four bases.
[0163] The eight masks used to synthesize the dinucleotide are
related to one another by translation or rotation. In fact, one
mask can be used in all eight steps if it is suitably rotated and
translated. For example, in the example above, a mask with a single
transparent region could be sequentially used to expose each of the
vertical columns, translated 90.degree., and then sequentially used
to allow exposure of the horizontal rows.
[0164] Tables 4 and 5 provide a simple computer program in Quick
Basic for planning a masking program and a sample output,
respectively, for the synthesis of a polymer chain of three
monomers ("residues") having three different monomers in the first
level, four different monomers in the second level, and five
different monomers in the third level in a striped pattern. The
output of the program is the number of cells, the number of
"stripes" (light regions) on each mask, and the amount of
translation required for each exposure of the mask.
4TABLE 4 Mask Strategy Program DEFINT A-Z DIM b(20), w(20), 1(500)
F$ = "LPT1:" OPEN f$ FOR OUTPUT AS #1 jmax = 3 `Number of residues
b(1) = 3: b(2) = 4: b(3) = 5 `Number of building blocks for res
1,2,3 g = 1: lmax(1) = 1 FOR j = 1 TO jmax: g= g * b(j): NEXT j
w(0) = 0: w(1) = g / b(1) PRINT #1, "MASK2.BAS ", DATE$, TIME$:
PRINT #1, PRINT #1, USING "Number of residues=##"; jmax FOR j = 1
TO jmax PRINT #1, USING " Residue ## ## building blocks"; j; b(j)
NEXT j PRINT #1, " PRINT #1, USING "Number of cells=####"; g: PRINT
#1, FOR j = 2 TO jmax lmax(j) = lmax(j - 1) * b(j - 1) w(j) = w(j -
1) / b(j) NEXT j FOR j = 1 TO jmax PRINT #1, USING "Mask for
residue ##"; j: PRINT #1, PRINT #1, USING " Number of stripes=###";
lmax(j) PRINT #1, USING " Width of each stripe=###"; w(j) FOR 1 = 1
TO lmax(j) a = 1 + (l - 1) * w(j - 1) ae = a + w(j) - 1 PRINT #1,
USING " Stripe ## begins at location ### and ends at ###"; l; a; ae
NEXT 1 PRINT #1, PRINT #1, USING " For each of ## building blocks,
translate mask by ## cell(s)"; b(j); w(j), PRINT #1, : PRINT #1, :
PRINT #1, NEXT j
[0165] .COPYRGT.Copyright 1990, Affymax N.V.
5TABLE 5 Masking Strategy Output Number of residues= 3 Residue 1 3
building blocks Residue 2 4 building blocks Residue 3 5 building
blocks Number of cells= 60 Mask for residue 1 Number of stripes= 1
Width of each stripe= 20 Stripe 1 begins at location 1 and ends at
20 For each of 3 building blocks, translate mask by 20 cell(s) Mask
for residue 2 Number of stripes= 3 Width of each stripe= 5 Stripe 1
begins at location 1 and ends at 5 Stripe 2 begins at location 21
and ends at 25 Stripe 3 begins at location 41 and ends at 45 For
each of 4 building blocks, translate mask by 5 cell(s) Mask for
residue 3 Number of stripes= 12 Width of each stripe= 1 Stripe 1
begins at location 1 and ends at 1 Stripe 2 begins at location 6
and ends at 6 Stripe 3 begins at location 11 and ends at 11 Stripe
4 begins at location 16 and ends at 16 Stripe 5 begins at location
21 and ends at 21 Stripe 6 begins at location 26 and ends at 26
Stripe 7 begins at location 31 and ends at 31 Stripe 8 begins at
location 36 and ends at 36 Stripe 9 begins at location 41 and ends
at 41 Stripe 10 begins at location 46 and ends at 46 Stripe 11
begins at location 51 and ends at 51 Stripe 12 begins at location
56 and ends at 56 For each of 5 building blocks, translate mask by
1 cell(s)
[0166] .COPYRGT.Copyright 1990, Affymax N.V.
[0167] V. Details of One Embodiment of a Fluorescent Detection
Device
[0168] FIG. 9 illustrates a fluorescent detection device for
detecting fluorescently labeled receptors on a substrate. A
substrate 112 is placed on an x/y translation table 202. In a
preferred embodiment the x/y translation table is a model no.
PM500-A1 manufactured by Newport Corporation. The x/y translation
table is connected to and controlled by an appropriately programmed
digital computer 204 which may be, for example, an appropriately
programmed IBM PC/AT or AT compatible computer. Of course, other
computer systems, special purpose hardware, or the like could
readily be substituted for the AT computer used herein for
illustration. Computer software for the translation and data
collection functions described herein can be provided based on
commercially available software including, for example, "Lab
Windows" licensed by National Instruments, which is incorporated
herein by reference for all purposes.
[0169] The substrate and x/y translation table are placed under a
microscope 206 which includes one or more objectives 208. Light
(about 488 nm) from a laser 210, which in some embodiments is a
model no. 2020-05 argon ion laser manufactured by Spectraphysics,
is directed at the substrate by a dichroic mirror 207 which passes
greater than about 520 nm light but reflects 488 nm light. Dichroic
mirror 207 may be, for example, a model no. FT510 manufactured by
Carl Zeiss. Light reflected from the mirror then enters the
microscope 206 which may be, for example, a model no. Axioscop 20
manufactured by Carl Zeiss. Fluorescein-marked materials on the
substrate will fluoresce >488 nm light, and the fluoresced light
will be collected by the microscope and passed through the mirror.
The fluorescent light from the substrate is then directed through a
wavelength filter 209 and, thereafter through an aperture plate
211. Wavelength filter 209 may be, for example, a model no. OG530
manufactured by Melles Griot and aperture plate 211 may be, for
example, a model no. 477352/477380 manufactured by Carl Zeiss.
[0170] The fluoresced light then enters a photomultiplier tube 212
which in some embodiments is a model no. R943-02 manufactured by
Hamamatsu, the signal is amplified in preamplifier 214 and photons
are counted by photon counter 216. The number-of photons is
recorded as a function of the location in the computer 204. Pre-Amp
214 may be, for example, a model no. SR440 manufactured by Stanford
Research Systems and photon counter 216 may be a model no. SR400
manufactured by Stanford Research Systems. The substrate is then
moved to a subsequent location and the process is repeated. In
preferred embodiments the data are acquired every 1 to 100 .mu.m
with a data collection diameter of about 0.8 to 10 .mu.m preferred.
In embodiments with sufficiently high fluorescence, a CCD detector
with broadfield illumination is utilized.
[0171] By counting the number of photons generated in a given area
in response to the laser, it is possible to determine where
fluorescent marked molecules are located on the substrate.
Consequently, for a slide which has a matrix of polypeptides, for
example, synthesized on the surface thereof, it is possible to
determine which of the polypeptides is complementary to a
fluorescently marked receptor.
[0172] According to preferred embodiments, the intensity and
duration of the light applied to the substrate is controlled by
varying the laser power and scan stage rate for improved
signal-to-noise ratio by maximizing fluorescence emission and
minimizing background noise.
[0173] While the detection apparatus has been illustrated primarily
herein with regard to the detection of marked receptors, the
invention will find application in other areas. For example, the
detection apparatus disclosed herein could be used in the fields of
catalysis, DNA or protein gel scanning, and the like.
[0174] VI. Determination of Relative Binding Strength of
Receptors
[0175] The signal-to-noise ratio of the present invention is
sufficiently high that not only can the presence or absence of a
receptor on a ligand be detected, but also the relative binding
affinity of receptors to a variety of sequences can be
determined.
[0176] In practice it is found that a receptor will bind to several
peptide sequences in an array, but will bind much more strongly to
some sequences than others. Strong binding affinity will be
evidenced herein by a strong fluorescent or radiographic signal
since many receptor molecules will bind in a region of a strongly
bound ligand. Conversely, a weak binding affinity will be evidenced
by a weak fluorescent or radiographic signal due to the relatively
small number of receptor molecules which bind in a particular
region of a substrate having a ligand with a weak binding affinity
for the receptor. Consequently, it becomes possible to determine
relative binding avidity (or affinity in the case of univalent
interactions) of a ligand herein by way of the intensity of a
fluorescent or radiographic signal in a region containing that
ligand.
[0177] Semiquantitative data on affinities might also be obtained
by varying washing conditions and concentrations of the receptor.
This would be done by comparison to known ligand receptor pairs,
for example.
[0178] VII. Examples
[0179] The following examples are provided to illustrate the
efficacy of the inventions herein. All operations were conducted at
about ambient temperatures and pressures unless indicated to the
contrary.
[0180] A. Slide Preparation
[0181] Before attachment of reactive groups it is preferred
to-clean the substrate which is, in a preferred embodiment a glass
substrate such as a microscope slide or cover slip. According to
one embodiment the slide is soaked in an alkaline bath consisting
of, for example, 1 liter of 95% ethanol with 120 ml of water and
120 grams of sodium hydroxide for 12 hours. The slides are then
washed under running water and allowed to air dry, and rinsed once
with a solution of 95% ethanol.
[0182] The slides are then aminated with, for example,
aminopropyltriethoxysilane for the purpose of attaching amino
groups to the glass surface on linker molecules, although any omega
functionalized silane could also be used for this purpose. In one
embodiment 0.1% aminopropyltriethoxysilane is utilized, although
solutions with concentrations from 10.sup.-7% to 10% may be used,
with about 10.sup.-3% to 2% preferred. A 0.1% mixture is prepared
by adding to 100 ml of a 95% ethanol/5% water mixture, 100
microliters (.mu.l) of aminopropyltriethoxysilane. The mixture is
agitated at about ambient temperature on a rotary shaker for about
5 minutes. 500 .mu.l of this mixture is then applied to the surface
of one side of each cleaned slide. After 4 minutes, the slides are
decanted of this solution and rinsed three times by dipping in, for
example, 100% ethanol.
[0183] After the plates dry, they are placed in a 110-120.degree.
C. vacuum oven for about 20 minutes, and then allowed to cure at
room temperature for about 12 hours in an argon environment. The
slides are then dipped into DMF (dimethylformamide) solution,
followed by a thorough washing with methylene chloride.
[0184] The aminated surface of the slide is then exposed to about
500 .mu.l of, for example, a 30 millimolar (mM) solution of
NVOC-GABA (gamma amino butyric acid) NHS (N-hydroxysuccinimide) in
DMF for attachment of a NVOC-GABA to each of the amino groups.
[0185] The surface is washed with, for example, DMF, methylene
chloride, and ethanol.
[0186] Any unreacted aminopropyl silane on the surface--that is,
those amino groups which have not had the NVOC-GABA attached--are
now capped with acetyl groups (to prevent further reaction) by
exposure to a 1:3 mixture of acetic anhydride in pyridine for 1
hour. Other materials which may perform this residual capping
function include trifluoroacetic anhydride, formicacetic anhydride,
or other reactive acylating agents. Finally, the slides are washed
again with DMF, methylene chloride, and ethanol.
[0187] B. Synthesis of Eight Trimers of "A" and "B"
[0188] FIG. 10 illustrates a possible synthesis of the eight
trimers of the two-monomer set: gly, phe (represented by "A" and
"B," respectively). A glass slide bearing silane groups terminating
in 6-nitro-veratryloxycarboxamide (NVOC-NH) residues is prepared as
a substrate. Active esters (pentafluorophenyl, OBt, etc.) of gly
and phe protected at the amino group with NVOC are prepared as
reagents. While not pertinent to this example, if side chain
protecting groups are required for the monomer set, these must not
be photoreactive at the wavelength of light used to protect the
primary chain.
[0189] For a monomer set of size n, n.times.l cycles are required
to synthesize all possible sequences of length l. A cycle consists
of:
[0190] 1. Irradiation through an appropriate mask to expose the
amino groups at the sites where the next residue is to be added,
with appropriate washes to remove the by-products of the
deprotection.
[0191] 2. Addition of a single activated and protected (with the
same photochemically-removable group) monomer, which will react
only at the sites addressed in step 1, with appropriate washes to
remove the excess reagent from the surface.
[0192] The above cycle is repeated for each member of the monomer
set until each location on the surface has been extended by one
residue in one embodiment. In other embodiments, several residues
are sequentially added at one location before moving on to the next
location. Cycle times will generally be limited by the coupling
reaction rate, now as short as 20 min in automated peptide
synthesizers. This step is optionally followed by addition of a
protecting group to stabilize the array for later testing. For some
types of polymers (e.g., peptides), a final deprotection of the
entire surface (removal of photoprotective side chain groups) may
be required.
[0193] More particularly, as shown in FIG. 10A, the glass 20 is
provided with regions 22, 24, 26, 28, 30, 32, 34, and 36. Regions
30, 32, 34, and 36 are masked, as shown in FIG. 10B and the glass
is irradiated and exposed to a reagent containg "A" (e.g., gly),
with the resulting structure shown in FIG. 10C. Thereafter, regions
22, 24, 26, and 28 are masked, the glass is irradiated (as shown in
FIG. 10D) and exposed to a reagent containing "B" (e.g., phe), with
the resulting structure shown in FIG. 10E. The process proceeds,
consecutively masking and exposing the sections as shown until the
structure shown in FIG. 10M is obtained. The glass is irradiated
and the terminal groups are, optionally, capped by acetylation. As
shown, all possible trimers of gly/phe are obtained.
[0194] In this example, no side chain protective group removal is
necessary. If it is desired, side chain deprotection may be
accomplished by treatment with ethanedithiol and trifluoroacetic
acid.
[0195] In general, the number of steps needed to obtain a
particular polymer chain is defined by:
n.times.l (1)
[0196] where:
[0197] n=the number of monomers in the basis set of monomers,
and
[0198] l=the number of monomer units in a polymer chain.
[0199] Conversely, the synthesized number of sequences of length l
will be:
n.sup.l. (2)
[0200] Of course, greater diversity is obtained by using masking
strategies which will also include the synthesis of polymers having
a length of less than l. If, in the extreme case, all polymers
having a length less than or equal to l are synthesized, the number
of polymers synthesized will be:
n.sup.l+n.sup.l-1+ . . . +n.sup.1. (3)
[0201] The maximum number of lithographic steps needed will
generally be n for each "layer" of monomers, i.e., the total number
of masks (and, therefore, the number of lithographic steps) needed
will be n.times.l. The size of the transparent mask regions will
vary in accordance with the area of the substrate available for
synthesis and the number of sequences to be formed. In general, the
size of the synthesis areas will be:
size of synthesis areas=(A)/(S)
[0202] where:
[0203] A is the total area available for synthesis; and
[0204] S is the number of sequences desired in the area.
[0205] It will be appreciated by those of skill in the art that the
above method could readily be used to simultaneously produce
thousands or millions of oligomers on a substrate using the
photolithographic techniques disclosed herein. Consequently, the
method results in the ability to practically test large numbers of,
for example, di, tri, tetra, penta, hexa, hepta, octapeptides,
dodecapeptides, or larger polypeptides (or correspondingly,
polynucleotides).
[0206] The above example has illustrated the method by way of a
manual example. It will of course be appreciated that automated or
semi-automated methods could be used. The substrate would be
mounted in a flow cell for automated addition and removal of
reagents, to minimize the volume of reagents needed, and to more
carefully control reaction conditions. Successive masks could be
applied manually or automatically.
[0207] C. Synthesis of a Dimer of an Aminopropyl Group and a
Fluorescent Group
[0208] In synthesizing the dimer of an aminopropyl group and a
fluorescent group, a functionalized durapore membrane was used as
substrate. The durapore membrane was a polyvinylidine difluoride
with aminopropyl groups. The aminopropyl groups were protected with
the DDZ group by reaction of the carbonyl chloride with the amino
groups, a reaction readily known to those of skill in the art. The
surface bearing these groups was placed in a solution of THF and
contacted with a mask bearing a checkerboard pattern of 1 mm opaque
and transparent regions. The mask was exposed to ultraviolet light
having a wavelength down to at least about 280 nm for about 5
minutes at ambient temperature, although a wide range of exposure
times and temperatures may be appropriate in various embodiments of
the invention. For example, in one embodiment, an exposure time of
between about 1 and 5000 seconds may be used at process
temperatures of between -70 and +50.degree. C.
[0209] In one preferred embodiment, exposure times of between about
1 and 500 seconds at about ambient pressure are used. In some
preferred embodiments, pressure above ambient is used to prevent
evaporation.
[0210] The surface of the membrane was then washed for about 1 hour
with a fluorescent label which included an active ester bound to a
chelate of a lanthanide. Wash times will vary over a wide range of
values from about a few minutes to a few hours. These materials
fluoresce in the red and the green visible region. After the
reaction with the active ester in the fluorophore was complete, the
locations in which the fluorophore was bound could be visualized by
exposing them to ultraviolet light and observing the red and the
green fluorescence. It was observed that the derivatized regions of
the substrate closely corresponded to the original pattern of the
mask.
[0211] D. Demonstration of Signal Capability
[0212] Signal detection capability was demonstrated using a
low-level standard fluorescent bead kit manufactured by Flow
cytometry Standarda and having model no. 824. This kit includes 5.8
.mu.m diameter beads, each impregnated with a known number of
fluorescein molecules.
[0213] One of the beads was placed in the illumination field on the
scan stage as shown in FIG. 9 in a field of a laser spot which was
initially shuttered. After being positioned in the illumination
field, the photon detection equipment was turned on. The laser beam
was unblocked and it interacted with the particle bead, which then
fluoresced. Fluorescence curves of beads impregnated with 7,000;
13,000; and 29,000 fluorescein molecules, are shown in FIGS. 11A,
11B, and 11C respectively. On each curve, traces for beads without
fluorescein molecules are also shown. These experiments were
performed with 488 nm excitation, with 100 .mu.W of laser power.
The light was focused through a 40 power 0.75 NA objective.
[0214] The fluorescence intensity in all cases started off at a
high value and then decreased exponentially. The fall-off in
intensity is due to photobleaching of the fluorescein molecules.
The traces of beads without fluorescein molecules are used for
background subtraction. The difference in the initial exponential
decay between labeled and nonlabeled beads is integrated to give
the total number of photon counts, and this number is related to
the number of molecules per bead. Therefore, it is possible to
deduce the number of photons per fluorescein molecule that can be
detected. For the curves illustrated in FIG. 11, this calculation
indicates the radiation of about 40 to 50 photons per fluorescein
molecule are detected.
[0215] E. Determination of the Number of Molecules Per Unit
Area
[0216] Aminopropylated glass microscope slides prepared according
to the methods discussed above were utilized in order to establish
the density of labeling of the slides. The free amino termini of
the slides were reacted with FITC (fluorescein isothiocyanate)
which forms a covalent linkage with the amino group. The slide is
then scanned to count the number of fluorescent photons generated
in a region which, using the estimated 40-50 photons per
fluorescent molecule, enables the calculation of the number of
molecules which are on the surface per unit area.
[0217] A slide with aminopropyl silane on its surface was immersed
in a 1 mM solution of FITC in DMF for 1 hour at about ambient
temperature. After reaction, the slide was washed twice with DMF
and then washed with ethanol, water, and then ethanol again. It was
then dried and stored in the dark until it was ready to be
examined.
[0218] Through the use of curves similar to those shown in FIG. 11,
and by integrating the fluorescent counts under the exponentially
decaying signal, the number of free amino groups on the surface
after derivitization was determined. It was determined that slides
with labeling densities of 1 fluoroscein per
10.sup.3.times.10.sup.3 to .about.2.times.2 nm could be
reproducibly made as the concentration of
aminopropyltriethoxysilane varied from 10.sup.-5% to
10.sup.-1%.
[0219] F. Removal of NVOC and Attachment of A Fluorescent
Marker
[0220] NVOC-GABA groups were attached as described above. The
entire surface of one slide was exposed to light so as to expose a
free amino group at the end of the gamma amino butyric acid. This
slide, and a duplicate which was not exposed, were then exposed to
fluorescein isothiocyanate (FITC).
[0221] FIG. 12A illustrates the slide which was not exposed to
light, but which was exposed to FITC. The units of the x axis are
time and the units of the y axis are counts. The trace contains a
certain amount of background fluorescence. The duplicate slide was
exposed to 350 nm broadband illumination for about 1 minute (12
mW/cm.sup.2, .about.350 nm illumination), washed and reacted with
FITC. The fluorescence curves for this slide are shown in FIG. 12B.
A large increase in the level of fluorescence is observed, which
indicates photolysis has exposed a number of amino groups on the
surface of the slides for attachment of a fluorescent marker. The
amino end of the spacer was deprotected and coupled to
NVOC-leucine. The entire slide was then illuminated with 12 mW of
325 nm broadband illumination. The slide was then coupled with
NVOC-phenylalanine and washed. The entire slide was again
illuminated, then coupled to NVOC-glycine and washed. The slide was
again illuminated and coupled to NVOC-glycine to form the sequence
shown in the last portion of FIG. 15A.
[0222] As shown in FIG. 15B, alternating regions of the slide were
then illuminated using a projection print using a 500.times.500
.mu.m checkerboard mask; thus, the amino group of glycine was
exposed only in the lighted areas. When the next coupling chemistry
step was carried out, NVOC-tyrosine was added, and it coupled only
at those spots which had received illumination. The entire slide
was then illuminated to remove all the NVOC groups, leaving a
checkerboard of YGGFL in the lighted areas and in the other areas,
GGFL. The Herz antibody (which recognizes the YGGFL, but not GGFL)
was then added, followed by goat anti-mouse fluorescein
conjugate.
[0223] The resulting fluorescence scan is shown in FIG. 15C, and
the color coding for the fluorescence intensity is again given on
the right. Dark areas contain the tetrapeptide GGFL, which is not
recognized by the Herz antibody (and thus there is no binding of
the goat anti-mouse antibody with fluorescein conjugated, and in
the red areas YGGFL is present. The YGGFL pentapeptide is
recognized by the Herz antibody and, therefore, there is antibody
in the lighted regions for the fluorescein-conjugated goat
anti-mouse to recognize.
[0224] Similar patterns are shown for a 50 .mu.m mask used in
direct contact ("proximity print") with the substrate in FIG. 15D.
Note that the pattern is more distinct and the corners of the
checkerboard pattern are touching when the mask is placed in direct
contact with the substrate (which reflects the increase in
resolution using this technique).
[0225] recirculated through the flow cell for 2 hours at room
temperature.
[0226] A first antibody, known as the Herz antibody, was applied to
the surface of the slide for 45 minutes at 2 .mu.g/ml in a
supercocktail (containing 1% BSA and 1% ovalbumin also in this
case). A second antibody, goat anti-mouse fluorescein conjugate,
was then added at 2 .mu.g/ml in the supercocktail buffer, and
allowed to incubate for 2 hours.
[0227] The results of this experiment are provided in FIG. 14.
Again, this figure illustrates fluorescence intensity as a function
of position. The fluorescence scale is shown on the right,
according to the color coding. This image was taken at 10 .mu.m
steps. This figure indicates that not only can deprotection be
carried out in a well defined pattern, but also that (1) the method
provides for successful coupling of peptides to the surface of the
substrate, (2) the surface of a bound peptide is available for
binding with an antibody, and (3) that the detection apparatus
capabilities are sufficient to detect binding of a receptor.
[0228] I. Monomer-by-Monomer Formation of YGGFL and Subsequent
Exposure to Labeled Antibody
[0229] Monomer-by-monomer synthesis of YGGFL and GGFL in alternate
squares was performed on a slide in a checkerboard pattern and the
resulting slide was exposed to the Herz antibody. This experiment
and the results thereof are illustrated in FIGS. 15A, 15B, 15C, and
15D.
[0230] In FIG. 15A, a slide is shown which is derivatized with the
aminopropyl group, protected in this case with t-BOC
(t-butoxycarbonyl). The slide was treated with TFA to remove the
t-BOC protecting group. E-aminocaproic acid, which was t-BOC
protected at its amino group, was then coupled onto the aminopropyl
groups. The aminocaproic acid serves as a spacer between the
aminopropyl group and the peptide to be synthesized.
[0231] G. Use of a Mask in Removal of NVOC
[0232] The next experiment was performed with a 0.1%
aminopropylated slide. Light from a Hg--Xe arc lamp was imaged onto
the substrate through a laser-ablated chrome-on-glass mask in
direct contact with the substrate.
[0233] This slide was illuminated for approximately 5 minutes, with
12 mW of 350 nm broadband light and then reacted with the 1 mM FITC
solution. It was put on the laser detection scanning stage and a
graph was plotted as a two-dimensional representation of position
color-coded for fluorescence intensity. The fluorescence intensity
(in counts) as a function of location is given on the color scale
to the right of FIG. 13A for a mask having 100.times.100 .mu.m
squares.
[0234] The experiment was repeated a number of times through
various masks. The fluorescence pattern for a 50 .mu.m mask is
illustrated in FIG. 13B, for a 20 .mu.m mask in FIG. 13C, and for a
10 .mu.m mask in FIG. 13D. The mask pattern is distinct down to at
least about 10 .mu.m squares using this lithographic technique.
[0235] H. Attachment of YGGFL and Subsequent Exposure to Herz
Antibody and Goat Antimouse
[0236] In order to establish that receptors to a particular
polypeptide sequence would bind to a surface-bound peptide and be
detected, Leu enkephalin was coupled to the surface and recognized
by an antibody. A slide was derivatized with 0.1% amino
propyl-triethoxysilane and protected with NVOC. A 500 .mu.m
checkerboard mask was used to expose the slide in a flow cell using
backside contact printing. The Leu enkephalin sequence
(H.sub.2N-tyrosine,glycine,glycine,phenylalanine,leucine-CO.sub.-
2H, otherwise referred to herein as YGGFL) was attached via its
carboxy end to the exposed amino groups on the surface of the
slide. The peptide was added in DMF solution with the BOP/HOBT/DIEA
coupling reagents and
[0237] J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL
[0238] A synthesis using a 50 .mu.m checkerboard mask similar to
that shown in FIG. 15 was conducted. However, P was added to the
GGFL sites on the substrate through an additional coupling step. P
was added by exposing protected GGFL to light through a mask, and
subsequence exposure to P in the manner set forth above. Therefore,
half of the regions on the substrate contained YGGFL and the
remaining half contained PGGFL.
[0239] The fluorescence plot for this experiment is provided in
FIG. 16. As shown, the regions are again readily discernable. This
experiment demonstrates that antibodies are able to recognize a
specific sequence and that the recognition is not
length-dependent.
[0240] K. Monomer-by-Monomer Synthesis of YGGFL and YPGGFL
[0241] In order to further demonstrate the operability of the
invention, a 50 .mu.m checkerboard pattern of alternating YGGFL and
YPGGFL was synthesized on a substrate using techniques like those
set forth above. The resulting fluorescence plot is provided in
FIG. 17. Again, it is seen that the antibody is clearly able to
recognize the YGGFL sequence and does not bind significantly at the
YPGGFL regions.
[0242] L. Synthesis of an Array of Sixteen Different Amino Acid
Sequences and Estimation of Relative Binding Affinity to Herz
Antibody
[0243] Using techniques similar to those set forth above, an array
of 16 different amino acid sequences (replicated four times) was
synthesized on each of two glass substrates. The sequences were
synthesized by attaching the sequence NVOC-GFL across the entire
surface of the slides. Using a series of masks, two layers of amino
acids were then selectively applied to the substrate. Each region
had dimensions of 0.25 cm.times.0.0625 cm. The first slide
contained amino acid sequences containing only L amino acids while
the second slide contained selected D amino acids. FIGS. 18A and
18B illustrate a map of the various regions on the first and second
slides, respectively. The patterns shown in FIGS. 18A and 18B were
duplicated four times on each slide. The slides were then exposed
to the Herz antibody and fluorescein-labeled goat anti-mouse.
[0244] FIG. 19 is a fluorescence plot of the first slide, which
contained only L amino acids. Red indicates strong binding (149,000
counts or more) while black indicates little or no binding of the
Herz antibody (20,000 counts or less). The bottom right-hand
portion of the slide appears "cut off" because the slide was broken
during processing. The sequence YGGFL is clearly most strongly
recognized. The sequences YAGFL and YSGFL also exhibit strong
recognition of the antibody. By contrast, most of the remaining
sequences show little or no binding. The four duplicate portions of
the slide are extremely consistent in the amount of binding shown
therein.
[0245] FIG. 20 is a fluorescence plot of the second slide. Again,
strongest binding is exhibited by the YGGFL sequence. Significant
binding is also detected to YaGFL, YsGFL, and YpGFL. The remaining
sequences show less binding with the antibody. Note the low binding
efficiency of the sequence yGGFL.
[0246] Table 6 lists the various sequences tested in order of
relative fluorescence, which provides information regarding
relative binding affinity.
6TABLE 6 Apparent Binding to Herz Ab L-a.a. Set D-a.a. Set YGGFL
YGGFL YAGFL YaGFL YSGFL YsGFL LGGFL YpGFL FGGFL fGGFL YPGFL yGGFL
LAGFL faGFL FAGFL wGGFL WGGFL yaGFL fpGFL waGFL
[0247] VIII. Illustrative Alternative Embodiment
[0248] According to an alternative embodiment of the invention, the
methods provide for attaching to the surface a caged binding member
which in its caged form has a relatively low affinity for other
potentially binding species, such as receptors and specific binding
substances. Such techniques are more fully described in copending
application Ser. No. 404,920, filed Sep. 8, 1989, and incorporated
herein by reference for all purposes.
[0249] According to this alternative embodiment, the invention
provides methods for forming predefined regions on a surface of a
solid support, wherein the predefined regions are capable of
immobilizing receptors. The methods make use of caged binding
members attached to the surface to enable selective activation of
the predefined regions. The caged binding members are liberated to
act as binding members ultimately capable of binding receptors upon
selective activation of the predefined regions. The activated
binding members are then used to immobilize specific molecules such
as receptors on the predefined region of the surface. The above
procedure is repeated at the same or different sites on the surface
so as to provide a surface prepared with a plurality of regions on
the surface containing, for example, the same or different
receptors. When receptors immobilized in this way have a
differential affinity for one or more ligands, screenings and
assays for the ligands can be conducted in the regions of the
surface containing the receptors.
[0250] The alternative embodiment may make use of novel caged
binding members attached to the substrate. Caged (unactivated)
members have a relatively low affinity for receptors of substances
that specifically bind to uncaged binding members when compared
with the corresponding affinities of activated binding members.
Thus, the binding members are protected from reaction until a
suitable source of energy is applied to the regions of the surface
desired to be activated. Upon application of a suitable energy
source, the caging groups labilize, thereby presenting the
activated binding member. A typical energy source will be
light.
[0251] Once the binding members on the surface are activated they
may be attached to a receptor. The receptor chosen may be a
monoclonal antibody, a nucleic acid sequence, a drug receptor, etc.
The receptor will usually, though not always, be prepared so as to
permit attaching it, directly or indirectly, to a binding member.
For example, a specific binding substance having a strong binding
affinity for the binding member and a strong affinity for the
receptor or a conjugate of the receptor may be used to act as a
bridge between binding members and receptors if desired. The method
uses a receptor prepared such that the receptor retains its
activity toward a particular ligand.
[0252] Preferably, the caged binding member attached to the solid
substrate will be a photoactivatable biotin complex, i.e., a biotin
molecule that has been chemically modified with photoactivatable
protecting groups so that it has a significantly reduced binding
affinity for avidin or avidin analogs than does natural biotin. In
a preferred embodiment, the protecting groups localized in a
predefined region of the surface will be removed upon application
of a suitable source of radiation to give binding members, that are
biotin or a functionally analogous compound having substantially
the same binding affinity for avidin or avidin analogs as does
biotin.
[0253] In another preferred embodiment, avidin or an avidin analog
is incubated with activated binding members on the surface until
the avidin binds strongly to the binding members. The avidin so
immobilized on predefined regions of the surface can then be
incubated with a desired receptor or conjugate of a desired
receptor. The receptor will preferably be biotinylated, e.g., a
biotinylated antibody, when avidin is immobilized on the predefined
regions of the surface. Alternatively, a preferred embodiment will
present an avidin/biotinylated receptor complex, which has been
previously prepared, to activated binding members on the
surface.
IX. Conclusion
[0254] The present inventions provide greatly improved methods and
apparatus for synthesis of polymers on substrates. It is to be
understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reviewing the above description.
By way of example, the invention has been described primarily with
reference to the use of photoremovable protective groups, but it
will be readily recognized by those of skill in the art that
sources of radiation other than light could also be used. For
example, in some embodiments it may be desirable to use protective
groups which are sensitive to electron beam irradiation, x-ray
irradiation, in combination with electron beam lithograph, or x-ray
lithography techniques. Alternatively, the group could be removed
by exposure to an electric current. The scope of the invention
should, therefore, be determined not with reference to the above
description, but should instead be determined with reference to the
appended claims, along with the full scope of equivalents to which
such claims are entitled.
* * * * *