U.S. patent application number 09/951972 was filed with the patent office on 2002-10-10 for ink jet method of spotting probe, probe array and indentification methods.
Invention is credited to Okamoto, Tadashi, Suzuki, Tomohiro, Yamamoto, Nobuko.
Application Number | 20020146715 09/951972 |
Document ID | / |
Family ID | 27328809 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146715 |
Kind Code |
A1 |
Okamoto, Tadashi ; et
al. |
October 10, 2002 |
Ink jet method of spotting probe, probe array and indentification
methods
Abstract
Provided is a method of spotting a probe densely and efficiently
on a surface of a solid support. A liquid containing a probe is
attached to a solid support as droplets to form spots containing
the probe on the solid support by an ink jet method.
Inventors: |
Okamoto, Tadashi;
(Yokohama-shi, JP) ; Yamamoto, Nobuko;
(Isehara-shi, JP) ; Suzuki, Tomohiro; (Atsugi-shi,
JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
27328809 |
Appl. No.: |
09/951972 |
Filed: |
September 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09951972 |
Sep 14, 2001 |
|
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09126851 |
Jul 31, 1998 |
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Current U.S.
Class: |
435/6.11 ;
427/2.11; 435/287.2 |
Current CPC
Class: |
B01J 2219/00432
20130101; C07H 21/00 20130101; B01J 2219/00497 20130101; B01J
2219/00596 20130101; C12N 15/11 20130101; C12Q 1/00 20130101; C40B
50/14 20130101; B01J 2219/00729 20130101; B01J 2219/00637 20130101;
B01L 2200/143 20130101; B01J 2219/00659 20130101; C12N 2310/351
20130101; B01J 2219/00274 20130101; B01J 2219/00725 20130101; B01J
2219/00378 20130101; C40B 40/10 20130101; B82Y 30/00 20130101; G01N
33/6845 20130101; B01J 2219/00722 20130101; B01J 2219/00621
20130101; B01J 2219/00317 20130101; C12Q 1/6837 20130101; B01J
2219/00619 20130101; G01N 2035/00158 20130101; B01J 2219/00702
20130101; C40B 30/04 20130101; B01J 2219/00315 20130101; C40B 60/14
20130101; B01J 2219/00677 20130101; G01N 33/54366 20130101; B01J
19/0046 20130101; B01J 2219/00612 20130101; B01L 2400/0442
20130101; B01L 3/0268 20130101; B01J 2219/00527 20130101; B01J
2219/00585 20130101; B01J 2219/00605 20130101; C12N 11/14 20130101;
C40B 40/06 20130101; G01N 33/68 20130101; B01J 2219/00626 20130101;
C07B 2200/11 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 427/2.11 |
International
Class: |
C12Q 001/68; B05D
003/00; C12M 001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 1997 |
JP |
9-207837 |
Oct 20, 1997 |
JP |
9-287046 |
Jul 24, 1998 |
JP |
10-209923 |
Claims
What is claimed is:
1. A method of spotting a probe which can bind specifically to a
target to a solid support comprising the steps of: supplying a
liquid containing a probe on a surface of a solid support by an ink
jet method and attaching the liquid, and forming a spot of probe on
the surface of the solid support.
2. The method of spotting according to claim 1 wherein the probe is
a single-stranded nucleic acid probe.
3. The method of spotting according to claim 2 wherein the
single-stranded nucleic acid probe includes a single-stranded DNA
probe.
4. The method of spotting according to claim 2 wherein the
single-stranded nucleic acid probe includes an RNA probe.
5. The method of spotting according to claim 2 wherein the
single-stranded nucleic acid probe includes a single-stranded PNA
probe.
6. The method of spotting according to claim 2 wherein the surface
of the solid support has a first functional group and the
single-stranded nucleic acid probe has a second functional group,
and the first and second functional groups react each other by
contact.
7. The method of spotting according to claim 6 wherein the first
functional group on the surface of the solid support is a maleimido
group and the second functional group of the single-stranded
nucleic acid probe is a thiol (SH) group.
8. The method of spotting according to claim 7 wherein the solid
support is a glass plate and the maleimido group is introduced by
introducing an amino group on the surface of the glass plate and
then reacting the amino group with N-(6-maleimidocaproyloxy)
succinimide.
9. The method of spotting according to claim 7 wherein the solid
support is a glass plate and the maleimido group is introduced by
introducing an amino group on the surface of the glass plate and
then reacting the amino group with succinimidyl-4-(maleimido
phenyl) butyrate.
10. The method of spotting according to claim 7 wherein the
maleimido group is reacted with the thiol group for at least 30
minutes.
11. The method of spotting according to claim 10 wherein the
single-stranded nucleic acid comprises a single-stranded PNA probe,
at the terminus of which the thiol group exists, and the maleimido
group is reacted with the thiol group for at least 2 hours.
12. The method of spotting according to claim 11 wherein the thiol
group at the terminus of the single-stranded PNA probe is
introduced by binding cysteine group to the N-terminus of the
single-stranded PNA probe.
13. The method of spotting according to claim 6 wherein the first
functional group on the surface of the solid support is an epoxy
group and the second functional group of the single-stranded
nucleic acid probe is an amino group.
14. The method of spotting according to claim 13 wherein the solid
support is a glass plate and the epoxy group is introduced by
applying a silane compound having an epoxy group in the molecule
thereof on the surface of the glass plate and reacting the compound
with the glass plate.
15. The method of spotting according to claim 13 wherein the epoxy
group is introduced by applying polyglycidyl methacrylate having an
epoxy group on the solid support.
16. The method of spotting according to claim 1 wherein the liquid
contains urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at
5-10 wt %, and an acetylene alcohol at 1 wt % of the liquid.
17. The method of spotting according to claim 16 wherein the
acetylene alcohol has a structure represented by the following
general formula (I): 6(wherein, R.sub.1, R.sub.2, R.sub.3, and
R.sub.4 represent an alkyl group, each m and n represent an
integral, and m=0 and n=0 or 1.ltoreq.m+n.ltoreq.30, and when
m+n=1, m or n is 0.)
18. The method of spotting according to claim 2 wherein a
concentration of the single-stranded nucleic acid probe in the
liquid is 0.05-500 .mu.M.
19. The method of spotting according to claim 18 wherein a
concentration of the single-stranded nucleic acid probe in the
liquid is 2-50 .mu.M.
20. The method of spotting according to claim 2 wherein a length of
the single-stranded nucleic acid probe is 2-5,000 bases.
21. The method of spotting according to claim 20 wherein a length
of the single-stranded nucleic acid probe is 2-60 bases.
22. The method of spotting according to claim 1 wherein the ink jet
method is a bubble jet method.
23. The method of spotting according to claim 1 wherein the probe
is an oligopeptide or a polypeptide with a specific amino acid
sequence.
24. The method of spotting according to claim 1 wherein the probe
is a protein.
25. The method of spotting according to claim 24 wherein the
protein is an antibody.
26. The method of spotting according to claim 24 wherein the
protein is an enzyme.
27. The method of spotting according to claim 1 wherein the probe
is an enzyme.
28. The method of spotting according to claim 1 wherein the liquid
is supplied so as to form independent spots in a density of 10,000
spots per square inch on the solid support.
29. The method of spotting according to claim 1 wherein the solid
support has a flat surface and homogenous surface properties.
30. The method of spotting according to claim 29 wherein the liquid
is supplied on the surface of the solid support so as to obtain a
distance between the adjacent spots not smaller than the maximum
width of the spot.
31. The method of spotting according to claim 30 wherein blocking
is performed on the surface of the solid support to prevent a
sample from attaching to the surface other than spots of the
surface of the solid support.
32. The method of spotting according to claim 31 wherein the
blocking is achieved by using bovine serum albumin.
33. The method of spotting according to claim 1 wherein the solid
support is partitioned by a matrix arranged in a pattern on the
surface, a plurality of wells whose bottom is the surface of the
solid support exposed in the pattern are provided, and the liquid
is supplied to the respective wells.
34. The method of spotting according to claim 33 wherein the solid
support is optically transparent and the matrix is opaque.
35. The method of spotting according to claim 33 wherein the matrix
comprises a resin.
36. The method of spotting according to claim 33 wherein the
surface of the matrix is hydrophobic.
37. The method of spotting according to claim 33 wherein the bottom
of the wells is hydrophilic.
38. The method of spotting according to claim 33 wherein the matrix
has a thickness of 1-20 .mu.m.
39. The method of spotting according to claim 33 wherein the wells
have a maximum width of 200 .mu.m.
40. The method of spotting according to claim 33 wherein the matrix
has a width 1/2-2 times the maximum width of the wells.
41. A probe array comprising a plurality of spots of a probe, the
spots being provided independently at a plurality of sites of a
surface of a solid support in a density of 10,000 spots per square
inch or higher.
42. The probe array according to claim 41 wherein the solid support
has a flat surface and homogenous surface properties.
43. The probe array according to claim 42 wherein the probe is a
single-stranded nucleic acid probe.
44. The probe array according to claim 43 wherein the
single-stranded nucleic acid probe includes a single-stranded DNA
probe.
45. The probe array according to claim 43 wherein the
single-stranded nucleic acid includes a single-stranded RNA
probe.
46. The probe array according to claim 43 wherein the
single-stranded nucleic acid includes a single-strand ed PNA
probe.
47. The probe array according to claim 43 wherein the
single-stranded nucleic acid is covalently bound to the surface of
the solid support by a reaction between a first functional group on
the surface of the solid surface and a second functional group of
the single-stranded nucleic acid probe.
48. The probe array according to claim 47 wherein the first
functional group on the surface of the solid support is a maleimido
group and the second functional group of the single-stranded
nucleic acid probe is a thiol (SH) group.
49. The probe array according to claim 48 wherein the
single-stranded nucleic acid probe is a single-stranded PNA probe
and contains a cysteine residue on an N-terminus side.
50. The probe array according to claim 47 wherein the first
functional group on the surface of the solid support is an epoxy
group and the second functional group of the single-stranded
nucleic acid probe is an amino group.
51. The probe array according to claim 42 wherein the spots are
formed by supplying a liquid containing the probe on the solid
support.
52. The probe array according to claim 42 wherein the probe is an
oligopeptide or a polypeptide with a specific amino acid
sequence.
53. The probe array according to claim 42 wherein the probe is a
protein.
54. The probe array according to claim 53 wherein the protein is an
antibody.
55. The probe array according to claim 53 wherein the protein is an
enzyme.
56. The probe array according to claim 42 wherein the probe is an
antigen.
57. The probe array according to claim 42 wherein a distance
between the adjacent spots is not smaller than a maximum width of
the spot.
58. The probe array according to claim 41 wherein the solid support
is partitioned by a matrix arranged in a pattern on the surface, a
plurality of wells whose bottom is the surface of the solid support
exposed in a pattern are provided, and the liquid is supplied to
the respective wells.
59. The probe array according to claim 58 wherein the probe is a
single-stranded nucleic acid probe.
60. The probe array according to claim 59 wherein the
single-stranded nucleic acid probe includes a single-stranded DNA
probe.
61. The probe array according to claim 59 wherein the
single-stranded nucleic acid includes a RNA probe.
62. The probe array according to claim 59 wherein the
single-stranded nucleic acid includes a single-stranded PNA
probe.
63. The probe array according to claim 62 wherein the
single-stranded nucleic acid is covalently bound to the surface of
the solid support by a reaction between the first functional group
of the surface of the solid surface and the second functional group
on the single-stranded nucleic acid probe.
64. The probe array according to claim 63 wherein the first
functional group on the surface of the solid support is a maleimido
group and the second functional group of the single-stranded
nucleic acid probe is a thiol (SH) group.
65. The probe array according to claim 64 wherein the
single-stranded nucleic acid probe is a single-stranded PNA probe
and contains a cysteine residue on an N-terminal side.
66. The probe array according to claim 63 wherein the first
functional group on the surface of the solid support is an epoxy
group and the second functional group of the single-stranded
nucleic acid probe is an amino group.
67. The probe array according to claim 58 wherein the spots are
formed by supplying a liquid containing a probe on the solid
support.
68. The probe array according to claim 58 wherein the probe is an
oligopeptide or a polypeptide with a specific amino acid
sequence.
69. The probe array according to claim 58 wherein the probe is a
protein.
70. The probe array according to claim 69 wherein the protein is an
antibody.
71. The probe array according to claim 69 wherein the protein is an
enzyme.
72. The probe array according to claim 58 wherein the probe is an
antigen.
73. The probe array according to claim 58 wherein the matrix is
opaque.
74. The probe array according to claim 73 wherein the solid support
is optically transparent.
75. The probe array according to claim 58 wherein the matrix
comprises a resin.
76. The probe array according to claim 58 wherein the probe is
attached only to the wells.
77. The probe array according to claim 58 wherein the matrix has a
thickness of 1-20 .mu.m.
78. The probe array according to claim 58 wherein the wells have a
maximum width of 200 .mu.m.
79. The probe array according to claim 58 wherein a distance
between the wells is 1/2-2 times the maximum width of the
wells.
80. The probe array according to claim 41 wherein the probe array
comprises at least 2 spots each of which comprises a different kind
of probe.
81. A method of manufacturing a probe array having a plurality of
spots arranged independently in a plurality of sites on a surface
of a solid support, the spots containing a probe which can bind
specifically to a target substance comprising a step of supplying a
liquid containing the probe and attaching the liquid to a
predetermined site on the surface of the solid support by means of
an ink jet method to form the spots.
82. The method of manufacturing according to claim 81 wherein the
probe is a single-stranded nucleic acid probe.
83. The method of manufacturing according to claim 82 wherein the
single-stranded nucleic acid probe is a single-stranded DNA
probe.
84. The method of manufacturing according to claim 82 wherein the
single-stranded nucleic acid probe is an RNA probe.
85. The method of manufacturing according to claim 82 wherein the
single-stranded nucleic acid probe is a single-stranded PNA
probe.
86. The method of manufacturing according to claim 82 wherein the
surface of the solid surface has a first functional group and the
single-stranded nucleic acid probe has a second functional group,
and the first and the second functional groups react each other by
contact.
87. The method of manufacturing according to claim 86 wherein the
first functional group on the surface of the solid support is a
maleimido group and the second functional group of the
single-stranded nucleic acid probe is a thiol (SH) group.
88. The method of manufacturing according to claim 87 wherein the
solid support is a glass plate and a maleimido group is introduced
by introducing an amino group on the surface of the glass plate and
then reacting the amino group with N-(6-maleimidocaproyloxy)
succinimide.
89. The method of manufacturing according to claim 87 wherein the
solid support is a glass plate and the maleimido group is
introduced by introducing an amino group on the surface of the
glass plate and then reacting the amino group with
succinimidyl-4-(maleimido phenyl) butyrate.
90. The method of manufacturing according to claim 87 wherein the
maleimido group is reacted with the thiol group for at least 30
minutes.
91. The method of manufacturing according to claim 90 wherein the
single-stranded nucleic acid is a single-stranded PNA probe, at the
terminus of which the thiol group exists, the maleimido group is
reacted with the thiol group for at least 2 hours.
92. The method of manufacturing according to claim 91 wherein the
thiol group at the terminus of the single-stranded PNA probe is
introduced by binding cysteine group to an N-terminal of the
single-stranded PNA probe.
93. The method of manufacturing according to claim 86 wherein the
first functional group on the surface of the solid support is an
epoxy group and the second functional group of the single-stranded
nucleic acid probe has is an amino group.
94. The method of manufacturing according to claim 93 wherein the
solid support is a glass plate and the epoxy group is introduced by
applying a silane compound having an epoxy group in the molecule
thereof on the surface of the glass plate and reacting the compound
with the glass plate.
95. The method of manufacturing according to claim 93 wherein the
epoxy group is introduced by applying polyglycidyl methacrylate
having an epoxy group on the solid support.
96. The method of manufacturing according to claim 93 wherein the
liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %,
thiodiglycol at 5-10 wt %, and an acetylene alcohol at 1 wt % of
the liquid.
97. The method of manufacturing according to claim 96 wherein the
acetylene alcohol has a structure represented by the following
general formula (I): 7(wherein, R.sub.1, R.sub.2, R.sub.3, and
R.sub.4 represent an alkyl group, each m and n represent an
integral, and m=0 and n=0, or 1.ltoreq.m+n.ltoreq.30, and when
m+n=1, m or n is 0.)
98. The method of manufacturing according to claim 82 wherein a
concentration of the single-stranded nucleic acid probe in the
liquid is 0.05-500 .mu.M.
99. The method of manufacturing according to claim 98 wherein the
concentration of the single-stranded nucleic acid probe in the
liquid is 2-50 .mu.M.
100. The method of manufacturing according to claim 82 wherein a
length of the single-stranded nucleic acid probe is 2-5,000
bases.
101. The method of manufacturing according to claim 100 wherein the
length of the single-stranded nucleic acid probe is 2-60 bases.
102. The method of manufacturing according to claim 81 wherein the
ink jet method is a bubble jet method.
103. The method of manufacturing according to claim 81 wherein the
liquid is supplied so as to form independent spots in a density of
10,000 spots per square inch on the solid support of higher.
104. The method of manufacturing according to claim 81 wherein the
probe is an oligopeptide or a polypeptide with a specific amino
acid sequence.
105. The method of manufacturing according to claim 81 wherein the
probe is a protein.
106. The method of manufacturing according to claim 105 wherein the
protein is an antibody.
107. The method of manufacturing according to claim 105 wherein the
protein is an enzyme.
108. The method of manufacturing according to claim 81 wherein the
probe is an antigen.
109. The method of manufacturing according to claim 81 wherein the
solid support has a flat surface and homogenous surface
properties.
110. The method of manufacturing according to claim 109 wherein
blocking is performed, following spotting of the probe to the solid
support, to prevent a sample from attaching to the surface other
than the spots.
111. The method of manufacturing according to claim 110 wherein the
blocking comprises a step of immersing the solid support to which
the spots have been formed in an aqueous solution of bovine serum
albumin.
112. The method of manufacturing according to claim 111 wherein a
concentration of the aqueous solution of bovine serum albumin is
0.1-5%.
113. The method of manufacturing according to claim 111 wherein the
solid support is immersed in an aqueous solution of bovine serum
albumin for at least 2 hours.
114. The method of manufacturing according to claim 109 wherein the
liquid is supplied on the surface of the solid support so as to
obtain a distance between the adjacent spots not smaller than a
maximum width of the spot.
115. The method of manufacturing according to claim 81 wherein the
solid support is partitioned by a matrix arranged in a pattern on
the surface, a plurality of wells whose bottom is the surface of
the solid support exposed in a pattern are provided, and the liquid
is supplied to the respective wells.
116. The method of manufacturing according to claim 115 wherein the
solid support is optically transparent and the matrix is
opaque.
117. The method of manufacturing according to claim 115 wherein the
matrix comprises a resin.
118. The method of manufacturing according to claim 115 wherein the
surface of the matrix is hydrophobic.
119. The method of manufacturing according to claim 115 wherein the
bottom of the wells is hydrophilic.
120. The method of manufacturing according to claim 115 wherein the
wells have a maximum width of 200 .mu.m.
121. The method of manufacturing according to claim 115 wherein the
matrix has a width 1/2-2 times the maximum width of the wells.
122. The method of manufacturing according to claim 115 wherein a
thickness of the matrix is 1-20 .mu.m.
123. The method of manufacturing according to claim 115 wherein the
matrix pattern is formed by photolithography.
124. The method of manufacturing according to claim 123 wherein the
photolithography comprises a step of forming a resin layer on the
surface of the solid support, forming a photoresist layer on the
resin layer, exposing the photoresist layer to light in a pattern
corresponding to the matrix pattern, and developing to form the
pattern of the photoresist on the resin layer; and a step of
patterning the resin layer using the pattern of the photoresist as
a mask and then removing the pattern of the photoresists.
125. The method of manufacturing according to claim 123 wherein the
photolithography comprises the steps of forming a photosensitive
resin layer on the surface of the solid support, exposing the
photosensitive resin layer to light in a pattern corresponding to
the matrix pattern, and developing.
126. The method of manufacturing according to claim 125 wherein the
photosensitive resin layer is selected from the group consisted of
a UV resist, a DEEP-UV resist, or an ultraviolet cure resin.
127. The method of manufacturing according to claim 126 wherein the
UV resist is selected from the group consisted of a cyclized
polyisoprene-aromatic bisazide resist, a phenol resin-aromatic
azide compound resist, or a novolak resin-diazonaphtoquinone
resist.
128. The method of manufacturing according to claim 126 wherein the
DEEP-UV resin is a radiolysable polymer resist or a dissolution
suppressant resist.
129. The method of manufacturing according to claim 128 wherein the
radiation decomposition polymer resist is at least one selected
from a group consisting of polymethyl methacrylate, polymethylene
sulfone, polyhexafluorobutyl methacrylate, polymethylisopropenyl
ketone, and poly-1-trimethylsilyl propyne bromide.
130. The method of manufacturing according to claim 128 wherein the
dissolution suppressant resist is o-nitrobenzyl cholate ester.
131. The method of manufacturing according to claim 126 wherein the
DEEP-UV resist is polyvinylphenol-3,3'-diazidediphenyl sulfone or
polyglycidyl polymethacrylate.
132. The method of manufacturing according to claim 125 wherein
water repellency of the matrix pattern formed by patterning of the
photosensitive resin layer is further improved by postbaking of the
matrix pattern.
133. The method of manufacturing according to claim 115 wherein a
first functional group which can form a covalent bond with a second
functional group of the probe is introduced on the surface of the
solid support prior to formation of the wells.
134. The method of manufacturing according to claim 115 wherein a
first functional group which can form a covalent bond with a second
functional group of the probe is introduced on the surface of the
solid support following formation of the wells.
135. The method of manufacturing according to claim 134 wherein a
solution containing a compound for introducing the first functional
group to the surface of the solid support is supplied to the
wells.
136. The method of manufacturing according to claim 135 wherein the
solution is supplied to the wells by means of the ink jet
method.
137. The method of manufacturing according to claim 136 wherein the
solution is a silane coupling agent containing a silane compound
having an epoxy group or an amino group in its molecule.
138. The method of manufacturing according to claim 136 wherein the
solution contains a compound which can react with an amino group on
a glass substrate to introduce a maleimido group on the glass
substrate.
139. The method of manufacturing according to claim 138 wherein the
compound is N-maleimidocaproyloxy succinimide or
succinimidyl-4-(maleimid- ophenyl) butyrate.
140. A method for detecting whether a target substance is contained
in a sample, comprising the steps of: providing a probe array
comprising a plurality of spots each containing a probe which
specifically binds to the target substance, the spots being
arranged independently on a solid support; contacting the sample
with each of the spots; and detecting presence or absence of a
reacted product between the target substance and the probe, wherein
the respective spots are formed by spotting a liquid containing the
probe on the solid support by an ink jet method.
141. The method according to claim 140 wherein the target substance
is a single-stranded nucleic acid having a first base sequence and
the probe is a single-stranded nucleic acid probe having a second
base sequence complementary to the first base sequence.
142. The method according to claim 141 wherein the single-stranded
nucleic acid probe is a single-stranded DNA probe.
143. The method according to claim 141 wherein the single-stranded
nucleic acid probe is an RNA probe.
144. The method according to claim 141 wherein the single-stranded
nucleic acid probe is a single-stranded PNA probe.
145. The method according to claim 141 wherein the surface of the
solid surface has a first functional group and the single-stranded
nucleic acid probe has a second functional group, respectively, and
the functional groups react each other by contact.
146. The method according to claim 145 wherein the first functional
group on the surface of the solid support is a maleimido group and
the second functional group of the single-stranded nucleic acid
probe is a thiol (SH) group.
147. The method according to claim 146 wherein the solid support is
a glass plate and the maleimido group is introduced by introducing
an amino group on the surface of the glass plate and then reacting
the amino group with N-(6-maleimidocaproyloxy) succinimide.
148. The method according to claim 146 wherein the solid support is
a glass plate and the maleimido group is introduced by introducing
an amino group on the surface of the glass plate and then reacting
the amino group with succinimidyl-4-(maleimidophenyl) butyrate.
149. The method according to claim 146 wherein the maleimido group
is reacted with the thiol group for at least 30 minutes.
150. The method according to claim 149 wherein the single-stranded
nucleic acid comprises a single-stranded PNA probe having a thiol
group on the terminus and the maleimido group is reacted with the
thiol group for at least 2 hours.
151. The method according to claim 146 wherein the thiol group at a
terminus of the single-stranded PNA probe is introduced by binding
cysteine group to an N-terminus of the single-stranded PNA
probe.
152. The method according to claim 145 wherein the first functional
group on the surface of the solid support is an epoxy group and the
second functional group of the single-stranded nucleic acid probe
is an amino group.
153. The method according to claim 152 wherein the solid support is
a glass plate and the epoxy group is introduced by applying a
silane compound having an epoxy group in the molecule thereof on
the surface of the glass plate and reacting the compound with the
glass plate.
154. The method according to claim 152 wherein the epoxy group is
introduced by applying polyglycidyl methacrylate having an epoxy
group on the solid support.
155. The method according to claim 141 wherein the liquid contains
urea at 5-10 wt %, glycerin at 5-10 wt %, thiodiglycol at 5-10 wt
%, and an acetylene alcohol at 1 wt % of the liquid.
156. The method according to claim 155 wherein the acetylene
alcohol has a structure represented by the following general
formula (I): 8(wherein, R.sub.1, R.sub.2, R.sub.3, and R.sub.4
represent an alkyl group, each m and n represent an integral, and
m=0 and n=0 or 1.ltoreq.m+n.ltoreq.30, and when m+n=1, m or n is
0.)
157. The method according to claim 155 wherein a concentration of
the single-stranded nucleic acid probe in the liquid is 0.05-500
.mu.M.
158. The method according to claim 157 wherein a concentration of
the single-stranded nucleic acid probe in the liquid is 2-50
.mu.M.
159. The method according to claim 155 wherein a length of the
single-stranded nucleic acid probe is 2-5,000 bases.
160. The method according to claim 159 wherein a length of the
single-stranded nucleic acid probe is 2-60 bases.
161. The method according to claim 141 wherein the ink jet method
is a bubble jet method.
162. The method according to claim 140 wherein the probe is an
oligopeptide or a polypeptide with a specific amino acid
sequence.
163. The method according to claim 140 wherein the probe is a
protein.
164. The method according to claim 163 wherein the protein is an
antibody.
165. The method according to claim 163 wherein the protein is an
enzyme.
166. The method according to claim 140 wherein the probe is an
antigen.
167. The method according to claim 140 wherein the liquid is
supplied so as to form independent spots in a density of 10,000
spots per square inch on the solid support.
168. The method according to claim 140 wherein the solid support
has a flat surface and homogenous surface properties.
169. The method according to claim 168 wherein the liquid is
supplied on the surface of the solid support so as to obtain a
distance between the adjacent spots not smaller than the maximum
width of the spots.
170. The method according to claim 168 wherein blocking is
performed on the surface of the solid support to prevent the sample
from attaching to the surface other than the spots of the surface
of the solid support.
171. The method according to claim 170 wherein blocking is achieved
by using bovine serum albumin.
172. The method according to claim 140 wherein the solid support is
partitioned by a matrix arranged in a pattern on the surface, a
plurality of wells whose bottom is the surface of the solid support
exposed in the pattern are provided, and the liquid is supplied to
the respective wells.
173. The method according to claim 172 wherein the solid support is
optically transparent and the matrix is opaque.
174. The method according to claim 172 wherein the matrix comprises
a resin.
175. The method according to claim 172 wherein the surface of the
matrix is hydrophobic.
176. The method according to claim 172 wherein the bottom of the
wells is hydrophilic.
177. The method according to claim 172 wherein the matrix has a
thickness of 1-20 .mu.m.
178. The method according to claim 172 wherein the wells have a
maximum width of 200 .mu.m.
179. The method according to claim 172 wherein the matrix has a
width 1/2-2 times the maximum width of the wells.
180. A method of identifying a structure of a target substance
contained in a sample comprising the steps of: preparing a probe
array provided with spots of a probe, the probe being able to bind
specifically to the target substance, on a surface of a solid
support; contacting the sample to the spots; and detecting binding
between the target substance and the probe.
181. The method of identification according to claim 180 wherein
the target substance is a single-stranded nucleic acid, the
structure to be identified is a base sequence of the
single-stranded nucleic acid as the target substance, the probe
array is provided with a plurality of spots each of which contains
single-stranded nucleic acids with different base sequences on a
solid support, at least one of the spots contain a single-stranded
nucleic acid with a base sequence complementary to that anticipated
for the single-stranded nucleic acid as the target substance, and
the plurality of spots are formed by attaching a liquid containing
the respective single-stranded nucleic acids on the solid support
by means of an ink jet method.
182. The method of identification according to claim 181 wherein
the single-stranded nucleic acid probe is a single-stranded DNA
probe.
183. The method of identification according to claim 181 wherein
the single-stranded nucleic acid probe is an RNA probe.
184. The method of identification according to claim 181 wherein
the single-stranded nucleic acid probe is a single-stranded PNA
probe.
185. The method of identification according to claim 181 wherein
the surface of the solid surface and the single-stranded nucleic
acid probe have a first and a second functional groups,
respectively, and the functional groups react each other by
contact.
186. The method of identification according to claim 181 wherein
the first functional group on the surface of the solid support is a
maleimido group and the second functional group of the
single-stranded nucleic acid probe is a thiol (SH) group.
187. The method of identification according to claim 186 wherein
the solid support is a glass plate and the maleimido group is
introduced by introducing an amino group on the surface of the
glass plate and then reacting the amino group with
N-(6-maleimidocaproyloxy) succinimide.
188. The method of identification according to claim 186 wherein
the solid support is a glass plate and the maleimido group is
introduced by introducing an amino group on the surface of the
glass plate and then reacting the amino group with
succinimidyl-4-(maleimido phenyl) butyrate.
189. The method of identification according to claim 186 wherein
the maleimido group is reacted with the thiol group for at least 30
minutes.
190. The method of identification according to claim 189 wherein
the single-stranded nucleic acid is a single-stranded PNA probe
having a thiol group on the terminus thereof and the maleimido
group is reacted with the thiol group for at least 2 hours.
191. The method of identification according to claim 186 wherein
the thiol group at the terminus of the single-stranded PNA probe is
introduced by binding cysteine group to an N-terminus of the
single-stranded PNA probe.
192. The method of identification according to claim 185 wherein
the first functional group on the surface of the solid support is
an epoxy group and the second functional group of the
single-stranded nucleic acid probe is an amino group.
193. The method of identification according to claim 192 wherein
the solid support is a glass plate and the epoxy group is
introduced by applying a silane compound having an epoxy group in
the molecule on the surface of the glass plate and reacting the
compound with the glass plate.
194. The method of identification according to claim 192 wherein
the epoxy group is introduced by applying polyglycidyl methacrylate
having an epoxy group on the solid support.
195. The method of identification according to claim 181 wherein
the liquid contains urea at 5-10 wt %, glycerin at 5-10 wt %,
thiodiglycol at 5-10 wt %, and acetylene alcohol at 1 wt % of the
liquid.
196. The method of identification according to claim 195 wherein
the acetylene alcohol has a structure represented by the following
general formula (I): 9(wherein, R.sub.1, R.sub.2, R.sub.3, and
R.sub.4 represent an alkyl group, each m and n represent an
integral, and m=0 and n=0 or 1.ltoreq.m+n.ltoreq.30, and when
m+n=1, m or n is 0.)
197. The method of identification according to claim 195 wherein a
concentration of the single-stranded nucleic acid probe in the
liquid is 0.05-500 .mu.M.
198. The method of identification according to claim 197 wherein
the concentration of the single-stranded nucleic acid probe in the
liquid is 2-50 .mu.M.
199. The method of identification according to claim 197 wherein a
length of the single-stranded nucleic acid probe is 2-5,000
bases.
200. The method of identification according to claim 199 wherein a
length of the single-stranded nucleic acid probe is 2-60 bases.
201. The method of identification according to claim 181 wherein
the ink jet method is a bubble jet method.
202. The method of identification according to claim 180 wherein
the probe is an oligopeptide or a polypeptide with a specific amino
acid sequence.
203. The method of identification according to claim 180 wherein
the probe is a protein.
204. The method of identification according to claim 203 wherein
the protein is an antibody.
205. The method of identification according to claim 203 wherein
the protein is an enzyme.
206. The method of identification according to claim 180 wherein
the probe is an antigen.
207. The method of identification according to claim 180 wherein
the liquid is supplied so as to form independent spots in a density
of 10,000 spots per square inch on the solid support.
208. The method of identification according to claim 180 wherein
the solid support has a flat surface and homogenous surface
properties.
209. The method of identification according to claim 208 wherein
the liquid is supplied on the surface of the solid support so as to
obtain a distance between the adjacent spots not smaller than the
maximum width of the spots.
210. The method of identification according to claim 208 wherein
blocking is performed on the surface of the solid support to
prevent the sample from attaching to the surface other than spots
of the surface of the solid support.
211. The method of identification according to claim 210 wherein
blocking is achieved by using bovine serum albumin.
212. The method of identification according to claim 180 wherein
the solid support is partitioned by a matrix arranged in a pattern
on the surface, a plurality of wells whose bottom is the surface of
the solid support exposed in the pattern are provided, and the
liquid is supplied to the respective wells.
213. The method of identification according to claim 212 wherein
the solid support is optically transparent and the matrix is
opaque.
214. The method of identification according to claim 212 wherein
the matrix comprises a resin.
215. The method of identification according to claim 212 wherein
the surface of the matrix is hydrophobic.
216. The method of identification according to claim 212 wherein
the bottom of the wells is hydrophilic.
217. The method of identification according to claim 212 wherein
the matrix has a thickness of 1-20 .mu.m.
218. The method of identification according to claim 212 wherein
the wells have a maximum width of 200 .mu.m.
219. The method of identification according to claim 212 wherein
the matrix has a width 1/2-2 times a maximum width of the wells.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method of spotting a
probe on a solid support, a probe array and a method of
manufacturing thereof, and a method of detecting a target
single-stranded (ss) nucleic acid and a method of identifying a
base sequence of a target ss nucleic acid using the probe
array.
[0003] 2. Related Background Art
[0004] As a method to determine a base sequence of a nucleic acid,
detect a target nucleic acid in a sample, and identify various
bacteria swiftly and accurately, proposed is the use of a probe
array where one or more substances which can bind specifically to a
target nucleic acid, so-called probes, are arranged on a solid
support at a large number of sites. As a general method of
manufacturing such probe arrays as described in EP No. 0373203B1,
(1) the nucleic acid probe is synthesized on a solid support or (2)
a previously synthesized probe is supplied onto a solid support.
U.S. Pat. No. 5,405,783 discloses the method (1) in detail.
Concerning the method (2), U.S. Pat. No. 5,601,980 and Science Vol.
270, p. 467 (1995) teach a method of arranging cDNA in an array by
using a micropipet.
[0005] In the above method (1), it is not necessary to synthesize a
nucleic acid probe in advance, since the nucleic acid probe is
synthesized directly on a solid support. However, it is difficult
to purify a probe nucleic acid synthesized on a solid support. The
accuracy in determining the base sequence of a nucleic acid and in
the detection of a target nucleic acid in a sample using a probe
array largely depends on the correctness of the base sequence of
the nucleic acid probe. For the method (1), therefore, further
improvement in accuracy of a nucleic acid probe is required in
order to manufacture a probe array of higher quality. In the method
(2), a step of synthesizing a nucleic acid probe is required prior
to the fixation of the nucleic acid probe on a solid support, but
the nucleic acid probe can be purified before binding the probe to
a solid support. For this reason, presently, the method (2) is
considered to be more preferable than the method (1) as a method of
manufacturing a probe array of high quality. However, the method
(2) has a problem in the method of spotting a nucleic acid probe
densely on a solid support. For example, when a probe array is used
to determine the base sequence of a nucleic acid, it is preferable
to arrange as many kinds of nucleic acid probes as possible on a
solid support. When mutations in a gene are to be detected
efficiently, it is preferable to arrange nucleic acid probes of
sequences corresponding to the respective mutations on a solid
support. In addition, when a target nucleic acid in a sample or to
gene mutations and deletions are detected, it is desirable that the
amount of the sample taken from a subject, specifically a blood
sample, is as small as possible. Thus, it is preferable that as
much information as possible on the base sequence is obtained using
a small sample amount. Considering these points, it is preferable
that, for example, 10,000 or more nucleic acid probe spots per
square inch are arranged in a probe array.
SUMMARY OF THE INVENTION
[0006] As the result of the research carried out by the inventors
to solve above-discussed problems, they have found that an ink jet
ejection method enables spotting of a probe in a markedly high
density and achieved the present invention.
[0007] It is an object of the present invention to provide a method
of spotting an extremely small amount of probe efficiently and
accurately on a solid support without damaging the probe.
[0008] It is another object of the present invention to provide a
probe array that can provide more information on nucleic acid more
accurately even using a small amount of sample.
[0009] It is still another object of the present invention to
provide a method of efficiently manufacturing a probe array, in
which a large number of probes are bound to a solid support,
without damaging the probes.
[0010] It is further another object of the present invention to
provide a method of efficiently detecting a target substance that
may be contained in a sample.
[0011] It is still other object of the present invention to provide
a method of identifying the structure of a target substance to
obtain information on the structure of the target substance even
from a small amount of sample.
[0012] According to one aspect of the present invention, there
provided is a method of spotting a probe which can bind
specifically to a target tot a solid support. The method comprises
a step of supplying a liquid containing a probe on a surface of a
solid support by an ink jet method and adhering the liquid on the
surface of the solid support. The use of the spotting method
according to the above embodiment allows accurate and efficient
provision of a probe on a solid support and efficient manufacturing
of a probe array.
[0013] According to another aspect of the present invention,
provided is a probe array comprised of a plurality of spots of a
probe, where the spots are provided independently at a plurality of
sites of the surface of a solid support in a density of 10,000
spots per square inch or higher. This probe array has spots in a
remarkably high density so that much information can be obtained
even from a small amount of sample.
[0014] According to further aspect of the present invention,
provided is a method of manufacturing a probe array having a
plurality of spots arranged independently in a plurality of sites
on a surface of a solid support, the spots containing a probe which
can bind specifically to a target substance comprising a step of
supplying a liquid containing the probe and attaching the liquid to
a predetermined site on the surface of the solid support by means
of an ink jet method. According to this embodiment, a probe array
comprising spots arranged in a high density can be efficiently
manufactured without damaging the probe.
[0015] According to further aspect of the present invention,
provided is a method of detecting a target substance by contacting
a sample with each spot of a probe array having a probe that can
bind specifically to a target substance that may be contained in a
sample as a plurality of independent spots on a solid support to
detect a reaction product of the target substance and the probe on
the solid support to detect the presence/absence of the target
substance in the sample wherein the respective spots are formed by
spotting a liquid containing the probe on the solid support by the
ink jet method. According to this embodiment, a target substance
can be detected efficiently.
[0016] According to further aspect of the present invention,
provided is a method of identifying a structure of a target
substance contained in a sample comprising:
[0017] a step of preparing a probe array provided with spots of a
probe, which can bind specifically to a specific substance, on a
surface of a solid support;
[0018] a step of contacting the sample to the spots; and
[0019] a step of detecting binding between the target substance and
the probe.
[0020] U.S. Pat. No. 5,601,980 states that it is inappropriate to
use a conventional ink jet method in spotting of a nucleic acid
probe. In lines 31-52 in the second column of U.S. Pat. No.
5,601,980, it is said that the use of the ink jet printer technique
in which a small amount of ink is ejected by pressure wave is
inappropriate, because the pressure wave for ejecting ink may lead
to a drastic rise in the ink temperature and damage the nucleic
acid probe and scattering of the ink upon ejection may lead to
contamination of adjacent probe spots. Considering this, U.S. Pat.
No. 5,601,980 discloses a method of manufacturing a probe array in
which a drop of a liquid containing a nucleic acid probe is formed
on a tip of a micropipet utilizing gas pressure, while monitoring
the size of the drop, application of pressure is terminated when
the drop becomes the predetermined size, and the drop is applied on
a solid support.
[0021] U.S. Pat. No. 5,474,796 discloses manufacturing of
oligonucleotide array by forming a matrix of hydrophobic and
hydrophilic parts on a solid support surface and ejecting four
nucleotides sequentially to the hydrophilic part by means of a
piezoelectric impulse jet pump apparatus and a method of
determination of the base sequence of a target nucleic acid using
the oligonucleotide array. However, these prior arts do not
disclose a method in which nucleic acid probes each having a base
sequence of a predetermined length is ejected in advance using an
ink jet technique to arrange the nucleic acid probes accurately and
densely.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a schematic view illustrating a method of
manufacturing a probe array using a bubble jet head;
[0023] FIG. 2 is a cross sectional view taken along the line 2-2 of
the bubble jet head of FIG. 1;
[0024] FIG. 3 shows a graph comparing a theoretical amount and an
actual recovery of a nucleic acid probe spotted on an aluminum
plate by the bubble jet method in Example 3;
[0025] FIG. 4 shows a graph comparing a theoretical amount and an
actual recovery of a nucleic acid probe spotted on an aluminum
plate by the bubble jet method in Example 4;
[0026] FIG. 5A is a schematic plan view of one embodiment of a
probe array of the present invention, and FIG. 5B is a cross
sectional view taken along the line 5B-5B in FIG. 5A; and
[0027] FIG. 6 is to explain a spotting method in Example 8.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Outline of Method of Manufacturing Probe Array
[0028] FIGS. 1 and 2 are schematic diagrams illustrating a method
of manufacturing a probe array, for example, a nucleic acid probe
array, according to one embodiment of the present invention. In
FIG. 1, there are shown a liquid supply system (nozzle) 101 which
ejectably retains a liquid containing a probe, for example, a
nucleic acid probe, as an ejection liquid, a solid support 103 (for
example, transparent glass plate, etc.) to which the nucleic acid
probe is to be bound, and bubble jet head 105, a kind of ink jet
heads, provided with a mechanism to apply heat energy to the liquid
and thus eject the liquid. 104 denotes a liquid containing a
nucleic acid probe ejected from the bubble jet head 105. FIG. 2 is
a cross sectional view taken substantially along the line 2-2 of
the bubble jet head 105 of FIG. 1. In FIG. 2, there are shown the
bubble jet head 105, a liquid 107 containing a nucleic acid probe
to be ejected, and a substrate part 117 with a heating member
applying ejection energy to the liquid. The substrate part 117
comprises a protective film 109 made of silicone oxide etc.,
electrodes 111-1 and 111-2 made of aluminum etc., an exothermic
resistance layer 113 made of nichrome etc., a heat accumulator
layer 115, and a base material 116 made of alumina etc., with good
heat radiating properties. A liquid 107 containing a nucleic acid
probe comes up to an ejection orifice (ejection outlet) 119 and
forms a meniscus 121 by the predetermined pressure. When electric
signals from the electrodes 111-1 and 111-2 are supplied, a region
shown by 123 (bubbling region) rapidly generates heat and a bubble
appears in the liquid 107 contacting the region 123. The meniscus
ejects at the pressure and the liquid 107 is ejected from the
orifice 119 to fly toward the surface of a solid support 103.
Although the ejectable amount of the liquid using a bubble jet head
of such a structure depends on the size of the nozzle, etc., it can
be controlled to be about 4-50 picoliters and is very useful as
means to arrange nucleic acid probes in high density.
Relation between Ejected Liquid and Solid Support
[0029] Diameter of Spots on Solid Support
[0030] In order to obtain the probe density as described above (for
example, 10,000 probe spots per square inch, upper limit being
about 1.times.10.sup.6) on a solid support, it is preferable that
the diameter of each spot is about 20-100 .mu.m, for example, and
that spots are independent each other. These spots are determined
by properties of a liquid ejected from a bubble jet head and
surface properties of the solid support to which the liquid is
attached.
[0031] Properties of Ejection Liquid
[0032] Any liquid can be used as an ejection liquid, provided that
the liquid can be ejected from a bubble jet head, the liquid
ejected from the head arrives at the desired positions on a solid
support, and the liquid does not damage the nucleic acid probe when
it is mixed with nucleic acid probe and it is ejected.
[0033] From a viewpoint of liquid properties to be ejected from a
bubble jet head, the liquid preferably has properties such as
viscosity of 1-15 cps and surface tension of 30 dyn/cm or higher.
When viscosity is 1-5 cps and surface tension is 30-50 dyn/cm, the
position of arrival on a solid support becomes significantly
accurate and it is especially suitable.
[0034] Then considering ink jet ejection properties of the liquid
and stability of a nucleic acid probe in the liquid and, at
ejection by a bubble jet, it is preferable to contain a nucleic
acid probe of 2-5,000 mer, especially 2-60 mer in a concentration
of 0.05-500 .mu.M, especially 2-50 .mu.M.
[0035] Composition of Liquid
[0036] Composition of a liquid to be ejected from a bubble jet head
is not particularly restricted, provided that it does not
substantially affect a nucleic acid probe when it is mixed with a
nucleic acid probe or when it is ejected from the bubble jet head
as described above, and a liquid composition normally ejectable to
a solid support using the bubble jet head satisfies preferable
conditions. However, a preferable liquid contains glycerin, urea,
thiodiglycol or ethyleneglycol, isopropyl alcohol, and an acetylene
alcohol shown by the following formula (I): 1
[0037] wherein R1, R2, R3 and R4 represent alkyl groups,
specifically straight or branched alkyl groups containing 1-4
carbons, m and n represent integers, and m=0 and n=0 or
1.ltoreq.m+n.ltoreq.30, and when m+n=1, m or n is zero.
[0038] More specifically, a liquid comprising 5-10 wt % of urea,
5-10 wt % of glycerin, 5-10 wt % of thiodiglycol, and 0.02-5 wt %,
more preferably 0.5-1 wt % of an acetylene alcohol shown by the
above formula (I) is preferably used.
[0039] When this liquid is used, spots obtained by ejecting the
liquid containing a nucleic acid probe from a bubble jet head and
attached on a solid support are round, and an area where the
ejected liquid is attached is restricted. Thus, even when a nucleic
acid probe is spotted densely, connection of the adjacent spots can
be effectively prevented. No degradation of the nucleic acid probe
spotted on a solid support is observed. However, the properties of
the liquid used in manufacturing a nucleic acid probe array
according to the present invention are not restricted to those
mentioned above. For example, when structures like wells are
provided on a solid support surface to prevent spreading of the
liquid applied on the solid support by a bubble jet head and mixing
with adjacent spots, a liquid of a viscosity and surface tension
out of the above range, and a nucleic acid probe of a base length
and concentration out of the above range can be used.
[0040] Kinds of Functional Groups of Solid Support and Nucleic
Acids
[0041] A method to securely bind the nucleic acid probe to the
solid support, as well as to effectively retain the applied spot of
a nucleic acid probe at a more defined position on the solid
support to prevent cross contamination between adjacent spots, one
can endow the probe and the solid support with functional groups
which can react each other.
[0042] SH Group and Maleimido Group
[0043] The combination use of the maleimido group and the thiol
(--SH) group can be mentioned as a preferable example. That is, by
binding a thiol (--SH) group to the terminus of a nucleic acid
probe and treating the solid support surface to have a maleimido
group, the thiol group in a nucleic acid probe when supplied to the
surface of the solid support reacts with the maleimido group of the
solid support to immobilize the nucleic acid probe on the support,
forming probe spots on the predetermined positions on the solid
support. Especially, when such a nucleic acid probe containing a
thiol group at the terminus is dissolved in a liquid of the
above-mentioned composition, and applied on a solid support surface
having maleimido groups by means of a bubble jet head, the nucleic
acid probe solution can form a very small spot on the solid
support. As a result, small spots of a nucleic acid probe can be
formed on the predetermined positions of the surface of the solid
support. In this case, it is not necessary to provide a
construction such as wells comprised of partly hydrophilic and
hydrophobic matrix on the surface of the solid support to prevent
connection between spots.
[0044] For example, when a liquid containing a nucleic acid probe
of 18 mer nucleotides at a concentration of 8 .mu.M and controlled
to have the viscosity and surface tension within the above ranges
was ejected from a nozzle (an amount of ejection about 24
picoliters) using a bubble jet printer (Product name: BJC620; Canon
Inc., modified to print on a flat plane) with a space between the
solid support and the nozzle tip of the bubble jet head set about
1.2-1.5 mm, spots of a diameter about 70-100 .mu.m could be formed
and no spots due to scattering when the ejected liquid hit the
surface of the solid support (referred to as satellite spots
hereinafter) were observed. Reaction between maleimido groups on
the solid support and SH groups at the terminus of the nucleic acid
probes is completed in about 30 minutes at room temperature
(25.degree. C.), although depending on the conditions of an ejected
liquid. The time required is shorter than that required when a
piezoelectric jet head is used to eject a liquid. Although the
reason is not known, it is considered that the temperature of the
nucleic acid probe solution in the bubble jet head is elevated
according to its base principle so that the efficiency of reaction
between a maleimido group and a thiol group is increased to shorten
the reaction time.
[0045] Incidentally, a thiol group tends to become unstable under
an alkaline or neutral conditions and a disulfide bond (--S--S--),
which gives a dimer, may be formed. In order to prevent the
disulfide bond formation and to accomplish an effective reaction
between a thiol group and a maleimido group, it is preferable to
add thiodiglycol to the ejection liquid.
[0046] In order to introduce maleimido group onto a solid support
surface, various methods can be employed. For example, when a glass
substrate is used as the solid support, maleimido group can be
incorporated onto the surface of the solid support by an
introduction of amino group onto the substrate and the following
reaction between the amino group and a reagent containing
N-(6-maleimidocaproyloxy)succinimide (EMCS reagent: Dojin Co.,
Ltd.). The amino group introduction onto the surface can be
conducted by reacting an aminosilane coupling agent with the glass
substrate.
[0047] Structural Formula of EMCS 2
[0048] A nucleic acid probe having a thiol group at the terminus
thereof can be obtained by synthesizing a nucleic acid using
5'-thiol-modifier C6 (Glen Research Co., Ltd.) as a reagent for the
5'-terminus on an automatic DNA synthesizer followed by usual
deprotection reaction and purification by high performance liquid
chromatography.
[0049] Amino Group and Epoxy Group
[0050] As functional groups used for immobilization other than the
above-mentioned combination of the thiol group and the maleimido
group, a combination of the epoxy group (on solid support) and the
amino group (nucleic acid probe terminus) may also be used. Epoxy
groups can be introduced onto a solid support surface, for example,
by applying polyglycidyl methacrylate having an epoxy group onto
the surface of a solid support of a resin, or by applying a silane
coupling agent having an epoxy group onto the surface of glass
solid support for reaction.
[0051] As explained above, when functional groups are introduced
into a solid support surface and a terminus of a ss-nucleic acid
probe to form covalent bonds, the nucleic acid probe is more firmly
fixed to the solid support. In addition, since the nucleic acid
probe always binds to the solid support at its terminus, the states
of the nucleic acid probe in each spot become homogeneous. As a
result, hybridization between the nucleic acid probes and target
nucleic acids occurs in uniform conditions, thus the detection of a
target nucleic acid and the identification of a base sequence with
further improved accuracy can be realized. When nucleic acid probes
having a functional group on each terminus are covalently bound to
a solid support, a probe array can be produced quantitatively
without differences in the amount of bound probe DNA due to
difference in sequence or length, compared with nucleic acid probes
fixed on a solid support by non-covalent bond (for example,
electrostatically, etc.). In addition, all parts of the nucleic
acid participate in hybridization reaction, efficiency of
hybridization can be markedly improved. In addition, a linker such
as alkylene groups of 1-7 carbons or ethylene glycol derivatives
can be present between the ss nucleic acid portion which hybridizes
with a target nucleic acid and the functional group for binding
with a solid support. When such a nucleic acid probe is bound to a
solid support, a predetermined space can be provided between the
surface of the solid support and the nucleic acid probe so that
efficacy of reaction between a nucleic acid probe and a target
nucleic acid can be expected to be improved further.
[0052] Manufacturing Method of Probe
[0053] One of the preferred embodiments of the probe
array-manufacturing method will now be explained. First, a liquid
containing 7.5 wt % of glycerin, 7.5 wt % of urea, 7.5% of
thiodiglycol, and 1 wt % of an acetylene alcohol shown by the above
general formula (I) (for example, Product Name: Acetylenol EH;
Kawaken Fine Chemical Co., Ltd.) is prepared. A ss nucleic acid
probe of a length of, for example, about 2-5,000 mer, especially,
about 2-60 mer, having a thiol group at the terminus is synthesized
using an automatic DNA synthesizer. Nest, the nucleic acid probe is
mixed in the above liquid at a concentration in a range of 0.05-500
.mu.M, especially 2-50 .mu.M, to produce a liquid to be ejected
having a viscosity of 1-15 cps, especially 1-5 cps, and surface
tension of 30 dyn/cm or higher, especially 30-50 dyn/cm. Then, this
ejection liquid is filled in a nozzle of a bubble jet head, for
example. Maleimido groups are introduced on a solid support surface
according to the above method. The solid support is placed so that
a distance between the surface of the solid support having
maleimido groups and the nozzle tip of the bubble jet head becomes
as close as about 1.2-1.5 mm, and the bubble jet head is driven to
eject the liquid. Here, as the ejection conditions, it is desirable
to set printing pattern so as not to allow the connection between
the spots on a solid support each other. When a bubble jet head of
which resolution is 360.times.720 dpi is used for spotting,
preferable conditions are that one liquid ejection is followed by
twice idle ejections in the 360 dpi direction and one liquid
ejection is followed by 5 times idle ejections in the 720 dpi
direction. These conditions can provide a space of about 100 .mu.m
between spots and sufficiently prevent contamination between
adjacent spots. Then, the solid support is stood, for example, in a
humid chamber, until a reaction between the maleimido groups on a
solid support and the thiol groups of nucleic acid probes in a
liquid proceeds and the nucleic acid probes are securely fixed on
the solid support. It is sufficient to leave it at room temperature
(about 25.degree. C.) for about 30 minutes as described above.
Then, the nucleic acid probes not reacted on the solid support are
washed away to obtain a nucleic acid probe array.
[0054] Now, in order to improve detection accuracy (S/N ratio) in,
for example, detection of a target nucleic acid using this nucleic
acid probe array, it is preferable to block the solid support
surface after the nucleic acid probes were fixed to the support to
prevent the surface areas not binding the nucleic acid probes from
reacting with a target nucleic acid, etc., contained in a sample.
Blocking can be performed by, for example, immersing the solid
support in a 2% aqueous bovine serum albumin solution for two hours
or decomposing maleimido groups not bound to the nucleic acid
probes on the surface of the solid support. For example, DTT
(dithiothreitol), .beta.-mercaptoethanol, etc. can be used.
However, in terms of an effect of preventing adsorption of target
DNA, an aqueous solution of bovine serum albumin is the most
suitable. This step of blocking may be performed, as required. For
example, this blocking step can be omitted, when a sample can be
supplied restrictively to the respective spots of the probe array
and any sample would not attach substantially to the parts other
than the probe spots. The blocking step can be omitted, also when
wells have been formed on the solid support beforehand, and parts
other than wells are processed to inhibit attachment of nucleic
acid probes.
[0055] The probe arrays manufactured by such a method may have a
plurality of spots containing the same nucleic acid probe or a
plurality of spots each containing a different nucleic acid probe,
depending on applications. The probe array in which the nucleic
acid probes are arranged at a high density prepared a mentioned
above, can then be used for the detection of a target ss nucleic
acid and the identification of a base sequence. For example, when a
target ss nucleic acid of a known base sequence which may be
present in a test sample is detected, a ss nucleic acid having a
base sequence complementary to that of the target nucleic acid is
used as the probe, and the probe array in which a plurality of
spots containing the probe are arranged on a solid support is
prepared. Each sample is supplied to each spot of the probe array,
and the probe array is left standing under conditions allowing
hybridization between the target nucleic acid and the probe, then
the presence/absence of hybrid in each spot is detected by a known
method such as fluorescent detection. This enables detection of the
presence/absence of the target substance in a sample. When a probe
array is used to identify a base sequence of a target ss nucleic
acid contained in a sample, a plurality of ss nucleic acids having
base sequences complementary to the presumed sequences of the
target nucleic acid are spotted as probes on the solid support.
Then, aliquots of the sample are supplied to the respective spots
and incubated under conditions allowing hybridization of the target
nucleic acid and the probe, and then the presence/absence of
hybridization at each spot is detected by a known method such as
the fluorescence method. This enables identification of a base
sequence of a target ss nucleic acid. As other applications of the
probe array according to the present invention, for example,
application to screening of specific base sequences recognized by
DNA binding protein and chemical substances having a property to
bind to DNA can be considered.
[0056] Kinds of Ink Jet Head
[0057] Although a constitution in which a nucleic acid probe is
applied to a solid support by means of a bubble jet head is solely
illustrated above, a piezoelectric jet head ejecting a liquid in a
nozzle by vibration pressure of piezoelectric elements can also be
used in the present invention. However, a bubble jet head is
suitably used in the present invention, since a binding reaction to
a solid support is completed in a short period of time and
secondary structure of DNA is unfolded by heat so that efficiency
of the subsequent hybridization reaction can be increased, as
described above.
[0058] In addition, an ink jet system having a plurality of heads
can be used to form a plurality of spots simultaneously on a solid
support so that two or more spots may contain different nucleic
acid probes.
[0059] PNA/DNA
[0060] The present invention has been illustrated using a nucleic
acid probe as an example of probes. Nucleic acid probes include
deoxyribonucleic acid (DNA) probes, ribonucleic acid (RNA) probes,
and peptide nucleic acid (PNA) probes. PNAs are synthetic
oligonucleotides in which four bases (adenine, guanine, thymine,
and cytosine) contained in DNA are bound to a peptide backbone, not
to a sugar-phosphate backbone as shown in the following formula
(II): PNA Structural Formula (II) 3
[0061] wherein "Base" represents any one of four bases (adenine,
guanine, thymine, and cytosine) contained in DNA, and p represents
a base length of the PNA. PNAs can be synthesized, for example, by
methods known as tBOC-type solid phase synthesis and Fmoc-type
solid phase synthesis. PNAs are more resistant to enzymes such as
nucleases and proteases as compared to natural oligonucleotides of
DNA and RNA, hardly or not cleaved enzymatically, and stable in the
serum. Due to the absence of the sugar moiety or phosphate groups,
PNAs are rarely affected by ionic strength of a buffer. Therefore,
it is not required to control a salt concentration, etc., when PNAs
are reacted with a target ss nucleic acid. In addition, due to the
absence of electrostatic repulsion, a hybrid between PNA and a
target ss nucleic acid is considered to be more heat-stable than
those between a DNA probe and a target ss nucleic acid and between
an RNA probe and a target ss nucleic acid. From these
characteristics, PNAs are expected as probes used for the detection
of a target nucleic acid and the determination of a base sequence.
The method of manufacturing a nucleic acid probe array according to
the present invention is effective also when a PNA probe is used as
a nucleic acid probe and can easily manufacture a PNA probe array
in which PNA probes are arranged densely and very accurately.
Specifically, for example, to increase the density of a probe array
by securing a PNA probe on restricted positions on a solid support,
as in the case of DNA probes and RNA probes, two kinds of
functional groups which can react each other into the terminus of a
PNA probe and a solid support surface are introduced respectively.
A preferred combination of reactive groups is, as mentioned above,
a combination of a thiol group (at the terminus of PNA) and a
maleimido group (a solid support surface). A thiol group can be
introduced at the terminus of PNA by, for example, introducing a
cysteine (CH(NH.sub.2)(COOH)CH.sub.2SH) group, etc., containing a
thiol group in the N-terminus (corresponding to the 5'-terminus of
DNA) of a PNA probe. A cysteine group can be introduced at the
N-terminus of a PNA probe by, for example, reacting the amino group
of the N-terminus of a PNA probe and the carboxyl group of
cysteine. Further, using a suitable linker such as those containing
an amino group and a carboxyl group such as
N.sub.2H(CH.sub.2).sub.2O(CH.sub.2).sub.2OCH.sub.2COOH, the amino
group at N-terminus of a PNA probe is reacted with the carboxyl
group of the linker and then the amino group of the linker is
reacted with the carboxyl group of cysteine so as to bind cysteine
to the PNA probe via the linker. When a binding group to a solid
support is introduced via a linker as mentioned above, a part of
PNA probe interactive with a target substance can be separated from
the solid support by a predetermined distance so that a further
improvement in hybridization efficiency is expected.
[0062] PNA may have lower water-solubility than DNA of the same
base length as the polymer length of the PNA. Thus, when a liquid
for ink jet ejection is prepared, it is preferable to dissolve PNA
in trifluoroacetic acid (for example, a 0.1 wt % aqueous solution
of trifluoroacetic acid) etc., in advance and then prepare an
ejection liquid of properties compatible to ink jet ejection using
various solvents mentioned above. In particular, prior dissolution
in trifluoroacetic acid can prevent the conversion of the terminal
cysteine residues to cystine due to the oxidation of thiol groups
of PNA. Thus it is preferable for further improvement in efficiency
of a reaction between the thiol group of PNA and the maleimido
group on a solid support surface. Although the reaction time of 30
min is sufficient for a reaction between a thiol group introduced
at the terminus of a DNA probe or an RNA probe and a maleimido
group on a solid support surface (when a bubble jet head is used),
it is preferable to proceed a reaction for about 2 hours in case of
PNA even using a bubble jet head.
[0063] In the present invention, probes are not limited to nucleic
acid probes, and include substances which can bind specifically to
a target substance in a sample to be detected or analyzed, for
example, ligands which can bind specifically to receptors,
receptors which can bind specifically to ligands, oligopeptides and
polypeptides which can bind to oligopeptides and polypeptides
having specific amino acid sequences, and proteins (for example,
antibodies, antigens, enzymes, etc.).
[0064] As mentioned above, according to the method of manufacturing
a probe array comprising a step of supplying a probe solution to a
solid support using an ink jet ejection process, a probe array can
be manufactured very easily. In particular, when functional groups
are introduced both in a nucleic acid probe and in a solid support
surface so as to form a covalent bond between them, adjacent spots
do not connect each other even when a solid support on which wells,
etc. have not been provided in advance, that is, a solid support
which is substantially flat and has homogenous surface properties
(water-wettability, etc.) is used. As a result, a nucleic acid
probe array in which spots of a nucleic acid probe are arranged
accurately and densely can be manufactured extremely efficiently
and at a low cost.
[0065] This description does not intend to exclude a solid support
provided with wells on the surface in the present invention. For
example, when opaque matrix pattern (referred to as a black (BM)
matrix hereinafter) is previously formed between wells to which a
probe solution is supplied, detection accuracy (SN ratio) can be
further improved in optical detection (for example, detection of
fluorescence) of hybridization between a probe and a target
substance. In addition, when a matrix whose surface has a low
affinity to a probe solution is provided between adjacent wells,
the probe solution can be smoothly supplied to desired wells, even
when the solution is supplied to somewhat offset positions during
supply of the probe solution to wells. To enjoy such an effect, it
can be used a solid support on the surface of which wells are
provided. A solid support with a matrix formed on its surface, a
manufacturing method thereof, and a method of using the solid
support according to this embodiment are described below.
[0066] FIG. 5A and 5B show examples of a probe array according to
this embodiment of the present invention. FIG. 5A is a plan view
and FIG. 5B is a cross sectional view taken along the line 5B-5B of
FIG. 5A. This probe array has a configuration in which a matrix
pattern 125 in a framework structure containing hollowed parts
(wells) 127 are arranged in a form of a matrix are formed on a
solid support 103. The wells 127 separated by the matrix pattern
125 (projecting part) are provided as through holes (bored parts)
in the matrix pattern, the side walls of the holes being formed by
projecting parts, and a surface of the solid support 103 is exposed
at the bottom 129. The exposed surface of the solid support 103
forms a surface which can bind to a probe, and probes (not shown)
are fixed to the predetermined wells.
[0067] Materials to form the matrix pattern are preferably those
which make the matrix pattern opaque, considering improvement in
detection sensitivity, S/N ratio, and reliability, when a reaction
product of a probe and a target substance is detected optically,
for example, by measuring florescence emitted from the reaction
product. As these materials, metals (chromium, aluminum, gold,
etc.) and black resins, etc., can be exemplified. As the black
resins, included are resins such as acrylic, polycarbonate,
polystyrene, polyethylene, polyimide, acrylic monomer, and urethane
acrylate and photosensitive resins such as photoresists containing
black dyes or pigments. As specific examples of photosensitive
resins, for example, UV resist, DEEP-UV resist, ultraviolet cure
resins can be used. As UV resists, negative resists such as
cyclized polyisoprene-aromatic bisazide resists, and phenol
resin-aromatic azide compound resists, and positive resists such as
novolak resin-diazonaphtoquinone resists can be mentioned. As
DEEP-UV resists, positive resists, for example, radiolytic polymer
resists such as polymethyl methacrylate, polymethylene sulfone,
polyhexafluorobutyl methacrylate, polymethylisopropenyl ketone, and
poly-1-trimethylsilyl propylene bromide and dissolution suppressant
resists such as o-nitrobenzyl ester cholate, and negative resists
such as polyvinylphenol-3,3'-diazidediphenyl sulfone and glycidyl
polymethacrylate can be mentioned.
[0068] As ultraviolet curing resins, polyester acrylate, epoxy
acrylate and urethane acrylate, etc., containing about 2-10 wt % of
one or more photopolymerization initiators selected from a group
consisting of benzophenone and its substituted derivatives, benzoin
and its substituted derivatives, acetophenone and its substituted
derivatives, and oxime compounds such benzyl, etc can be
mentioned.
[0069] As black pigments, carbon black and black organic pigments
can be used.
[0070] When the reaction product of a probe and a target substance
is not detected optically and when light from a matrix does not
affect optical detection of a reaction product, the use of
non-light-shielding substances as a material for a matrix pattern
is not excluded.
[0071] As one of the methods of forming a matrix pattern using the
above materials, a method in which a photoresist layer is formed on
a resin or a metal layer formed on the surface of a substrate, and
after the patterning of the resist layer, the resin is patterned by
a process such as etching. When a photosensitive resin is used, the
resin itself can be exposed, developed, and cured if required, by a
process of photolithography using a photomask for patterning. When
a matrix 125 is made of a resin, the surface of the matrix 125 is
hydrophobic. This configuration is preferable when an aqueous
solution is used as a solution containing a probe and supplied to
wells. That is, when a probe solution is supplied to wells by the
ink jet method, the probe solution can be supplied very smoothly to
desired wells, even when the probe solution is supplied in slightly
offset positions. In addition, when different probes are supplied
to adjacent wells simultaneously, cross-contamination of these
different probe solutions supplied to the wells can be
prevented.
[0072] Since a solution of a probe, a biomaterial, such as peptides
and nucleic acids, is often an aqueous solution, this constitution
in which a matrix pattern is water-repellent can be suitably used
in such occasions.
[0073] Next, a method of making a bottom of a well (an exposed part
of a solid support surface) which can bind a probe is described. A
functional group to be retained on the bottom of a well is
determined by the functional group to be carried on a probe. For
example, when a nucleic acid probe in which a thiol group is
introduced at the terminus is used, previous introduction of a
maleimido group to a solid support surface, as mentioned above,
makes the thiol group of the nucleic acid probe supplied to wells
form a covalent bond with the maleimido group on the surface of the
solid support and the nucleic acid probe is then fixed on the
surface of the solid support. Similarly, with a nucleic acid probe
having an amino group at the terminus, it is preferable to
introduce epoxy groups to a solid support surface. As other
combinations of these functional groups, for example, a combination
of a carboxyl group for a nucleic acid probe (by introducing a
succinimide derivative to the terminus of a nucleic acid probe) and
an amino group for a solid support surface is preferable. This
combination of amino and epoxy groups is inferior in immobilization
of the ink jet-ejected nucleic acid probe on a solid support to a
combination of thiol and maleimido groups but to a negligible
extent when wells are provided on the solid support.
[0074] The amino or epoxy group can be introduced to a glass plate
as the solid support by, first treating the surface of the glass
plate with an alkali solution such as potassium hydroxide and
sodium hydroxide to expose hydroxyl groups (silanol groups) to the
surface, and then reacting a silane coupling agent containing a
silane compound to which an amino group has been introduced (for
example, N-.beta.-(aminoethyl)-.gamma.-ami-
nopropyltrimethoxysilane, etc.) or a silane compound to which an
epoxy group has been introduced (for example,
.gamma.-glycidoxypropyltrimethoxy- silane, etc.) with a hydroxyl
group of the surface of the glass plate. To introduce maleimido
groups to the surface of the glass plate, the amino groups
introduced by the above method are reacted with
N-maleimidocaproyloxy succinimide or succinimidyl-4-(maleimido
phenyl)butyrate, etc.
[0075] The structures of
N-.beta.-(aminoethyl)-.gamma.-aminopropyltrimetho- xysilane,
.gamma.-glycidoxypropyltrimethoxysilane, and
succinimidyl-4-(maleimido phenyl)butyrate are shown below:
[0076] N-.beta.-(aminoethyl)-.gamma.-aminopropyltrimethoxysilane
(CH.sub.3O).sub.2SiC.sub.3H.sub.6NHC.sub.2H.sub.4NH.sub.2
[0077] .gamma.-glycidoxypropyltrimethoxysilane 4
[0078] (CH.sub.3O).sub.3SiC.sub.3H.sub.6OCH.sub.2
[0079] Succinimidyl-4-(maleimido phenyl)butyrate 5
[0080] When an epoxy group is introduced to a solid support surface
in the above surface treatment of a solid support, the base of
wells can be made hydrophilic after binding the epoxy groups to a
probe, by opening unreacted epoxy rings using an aqueous solution
of ethanol amine, etc., to change them into hydroxyl groups. This
operation is preferable, when an aqueous solvent containing a
target substance that will react specifically to a probe is
supplied to wells to which the probe has been bound.
[0081] When a resin plate is used as a solid support, hydroxyl
groups, carboxyl groups, or amino groups can be introduced to the
surface of resin substrate according to the method described in
Chapter 5 of "Organic Thin Films and Surface", Vol. 20, Academic
Press. Alternatively, after introducing hydroxyl groups by this
method, as is shown for the glass plate mentioned above, amino
groups or epoxy groups can be introduced by using a silane compound
having amino group or epoxy group. Further a maleimido group can be
introduced. Functional groups can be introduced either before or
after the matrix pattern is formed on a solid support. Before
matrix pattern formation, a reaction solution required for
introduction of a functional group can be supplied to a solid
support surface by spin coating or dip coating, etc. After matrix
formation, a reaction solution can be supplied to each well by the
ink jet method, etc.
[0082] To bind a probe to a resin substrate, for example, hydroxyl
groups are introduced by oxidation of the surface of a resin
substrate, then the hydroxyl groups are reacted with a silane
coupling agent comprised of a silane compound containing an amino
group to introduce amino groups, and each amino group is reacted
with a functional group of a probe, as described in Japanese Patent
Application Laid-Open No. 60-015560.
[0083] When the substrate after treatment is hydrophilic,
above-mentioned resins to make matrix pattern formation can be used
without any treatment as a relatively water-repellent material.
When further repellency is required, a water-repellant can be added
to a matrix material. When a matrix pattern is formed from a
photosensitive resin such as photoresists, post-baking under
appropriate conditions following exposure and development can
provide stronger repellency to the matrix pattern.
[0084] When a probe solution is lipophilic, although it has been
explained mainly on a hydrophilic probe solution, treatment can be
performed in opposite.
[0085] The size and shape of wells in a matrix pattern can be
selected according to the size of a substrate, the size of an array
as a whole finally prepared, the number and a type of probe
constituting the array, or a method of forming a matrix pattern, a
method of supplying a probe solution to wells in matrix pattern,
and a method of detection, etc.
[0086] Cross section of wells by a plane parallel to the substrate
can be various shapes, in addition to squares as shown in FIG. 5,
such as rectangles, various polygons, circles, and ovals.
[0087] Preferably, wells have a maximum width of 300 .mu.m or less,
considering the number of reactants and a size of a whole array.
For example, as shown in FIG. 5, when a cross section taken
parallel to a substrate is square, one side can be 200 .mu.m or
less in length. Preferably, when wells are rectangular, the maximum
side is 200 .mu.m or less, and when wells are round, the diameter
is 200 .mu.m or less. The minimum limit in length is about 1
.mu.m.
[0088] Wells can be arranged in various patterns as required. Wells
can be arranged at equal intervals making rows and columns as shown
in FIG. 5, or wells can be arranged so as to shift from the
positions of wells in adjacent lines.
[0089] A distance between adjacent wells is preferably set not to
cause cross-contamination even when the ejection positions are
somewhat offset from the position of the target well to which a
probe solution is supplied by, for example, the ink jet method. In
addition, considering a size of a whole array, cross-contamination,
and handling properties in supply of various solutions, the
distance between the adjacent wells is in the range of 1/2 to 2
times the maximum width.
[0090] For example, it is desirable 100.times.100 or
1,000.times.1,000 or more types of probes are present in a probe
array for fully displaying functions of combinatorial chemistry,
and the size of a substrate is desirably 1 inch.times.1 inch or 1
cm.times.1 cm, to be suitable for automation of operations such as
probe fixation, sample supply and detection, thus for square wells
it is preferable to set a side of a square of a well at 1-200 .mu.m
or less and a distance between adjacent wells is at 200 .mu.m or
less, considering the matrix size.
[0091] The thickness of a matrix (height from the solid support
surface) is determined considering a method of forming the matrix
pattern, volume of wells, and volume of a probe solution supplied.
It is preferably 1-20 .mu.m. Such a thickness enables, when a probe
solution is supplied to each well by the ink jet ejection method,
to retain the probe solution at predetermined positions on a solid
support and to prevent cross-contamination very efficiently, even
when the properties of the probe solution should be not suitable
for forming small spots on the solid support surface, in relation
to the conditions for the ink jet ejection method.
[0092] When a well has a size of the upper limits of the
above-described desirable ranges, that is, 200 .mu.m.times.200
.mu.m.times.20 .mu.m, the well volume is 800 pl. When this size is
used and a distance between adjacent wells (x in FIG. 1) is also
set at 200 .mu.m, a density of wells of 625 wells/cm.sup.2 is
obtained. That is, an array with a well density of an order of
10.sup.2 wells/cm.sup.2 or more can be obtained. When a well is a
square with a side of 5 .mu.m, a distance between adjacent wells is
set at 5 .mu.m, and a thickness of the matrix pattern is set at 4
.mu.m, a volume of a well is 0.1 pl and the density of wells is
1,000,000 wells/cm.sup.2. Since patterning of 5 .mu.m.times.5
.mu.m.times.4 .mu.m is possible in the present fine processing
technology, an array with a well density of an order of 106
wells/cm.sup.2 or more can be included in the scope of the present
invention.
[0093] In this embodiment, the feeding volume of a probe solution
or a substance to be reacted with a probe supplied to a well is 0.1
picoliters (pl) to 1 nanoliter (nl) from the above calculation,
when the volume to be supplied is deemed to be the same as or
almost the same as the volume of the well. When a matrix has little
affinity to a solution to be supplied, it is possible to supply the
solution in an volume exceeding the well volume which is retained
above the opening of the well due to surface tension, depending to
the type of the solution. In such a case, for example, a solution
in a volume 10 to several tens of times larger than that of the
well can be supplied and retained. That is, several picoliters to
several tens of nanoliters of a solution are supplied. In any
cases, a probe solution is preferably supplied to wells using the
ink jet method that can supply such a small amount of solution with
position accuracy and supply accuracy, although microdispensers and
micropipettes can also be used. In the ink jet printing, an ink is
ejected with positioning at high accuracy of an order of .mu.m.
This method is thus quite suitable for supplying a solution to
wells. Since a volume of ink to be ejected is several tens of
picoliters to several nanoliters, the ink jet method can be said to
be suitable for supplying a solution, also in this respect.
[0094] According to this embodiment, spreading of droplets can be
controlled quantitatively by the reaction between a nucleic acid
probe and a solid support surface as well as by wells. In addition,
even when a liquid is ejected in a somewhat offset direction, when
a droplet lands on an area containing a well, the droplet part on
the matrix is repelled and drawn into the well smoothly, since the
matrix has no affinity to the ejected solution.
[0095] The ink jet method used in the present invention is not
particularly restricted, and a piezo jet method, a bubble jet
method utilizing thermal bubbling, etc., can be employed.
[0096] Any materials can be used as the solid support 103 according
to one embodiment of the present invention, so long as various
functional groups as described above can be introduced to the
surface. According to the second embodiment of the present
invention, preferred materials are those on the surface of which a
matrix pattern can be formed. When the reaction product of a probe
and a target substance is detected optically by a detection system
via a solid support, the solid support is preferably transparent.
As these materials, glass including synthetic quartz and fused
quartz, silicone, acrylic resins, polycarbonate resins, polystyrene
resins, and vinyl chloride resins, etc. can be mentioned. When the
reaction product is detected optically not via a solid support, it
is preferable to use an optically black solid support, and resin
substrates containing black dyes or pigments such as carbon black
are used.
[0097] In the present invention, a solution which may contain a
substance which reacts with the probe (a test solution) is supplied
to a probe array and left under suitable reaction conditions to
proceed the reaction. When plural test solutions must be supplied
to the array, at least one test solution is supplied to plural
wells in the probe array, respectively. In this case, as shown
above, when the supplied solution has an affinity to wells
containing a fixed probe in the already formed probe array and has
no affinity to a matrix pattern, quantitative supply of the
solution to a restricted supply area can be achieved without
cross-contamination. Since most of biomaterials are water-soluble,
wells are hydrophilic and a matrix pattern is water-repellent. In
addition, the use of the ink jet method in supply of these
substances for reaction as shown above can quantitatively supply a
very small amount of solution.
[0098] According to the present invention, very small amounts of a
probe solution and a test solution are used. Thus, it is desirable
to include conditions for preventing evaporation or vaporization of
the supplied solutions for both cases.
[0099] The present invention is described in more detail referring
to the following examples.
EXAMPLE 1
[0100] Manufacturing of Nucleic Acid Probe Array Using Bubble Jet
Printer and Evaluation of the Probe Array
[0101] (1) Washing of Substrate
[0102] A glass plate of 1 inch.times.1 inch was placed in a rack
and immersed in an ultrasonic washing detergent overnight. After
ultrasonic washing in the detergent for 20 minutes, the detergent
was removed by rinsing with water. After rinsing with distilled
water, ultrasonic treatment was performed in a container containing
distilled water for 20 minutes. The glass plate was immersed for 10
minutes in a 1 N sodium hydroxide solution preheated to 80.degree.
C. Then, the plate was washed with water and distilled water to
prepare a glass plate for a probe array.
[0103] (2) Surface Treatment
[0104] A 1 wt % aqueous solution of a silane coupling agent
(Product name: KBM603; Shin-Etsu Chemical Co., Ltd.) containing a
silane compound having an amino group
(N-.beta.-(aminoethyl)-.gamma.-aminopropyltrimethoxysilane- ) was
stirred at room temperature for 2 hours to hydrolyze methoxy groups
of the above silane compound. Then, the substrate was immersed in
this solution at room temperature (25.degree. C.) for 20 minutes,
drawn up from the solution, and dried by blowing nitrogen gas to
both sides of the glass plate. Then, the glass plate was baked for
1 hour in an oven heated to 120.degree. C. to complete silane
coupling treatment to introduce an amino group on the surface of
the substrate. Then, 2.7 mg of N-(6-maleimidocaproyloxy)
succinimide (Dojin Co., Ltd.) (abbreviated as EMCS hereinafter) was
weighed and dissolved in a mixture of DMSO/ethanol (1:1) to a final
concentration of 0.3 mg/ml to prepare an EMCS solution. The glass
plate subjected to silane coupling treatment was immersed in the
EMCS solution at room temperature for 2 hours for the reaction of
the amino groups carried on the surface of the glass plate by
silane coupling treatment and the carboxyl groups of the EMCS
solution. In this condition, the glass plate obtained maleimido
groups derived from EMCS on its surface. The glass plate drawn up
from the EMCS solution was washed successively with a mixed solvent
of dimethylsulfoxide and ethanol and with ethanol and then dried
under a nitrogen gas atmosphere.
[0105] (3) Synthesis of DNA Probe
[0106] A single-stranded (ss) nucleic acid of SEQ ID No. 1 was
synthesized using an automatic DNA synthesizer. A thiol (--SH)
group was introduced at the terminus of the ss DNA of SEQ ID No. 1
using Thiol-Modifier (Glen Research Co., Ltd.) during synthesis by
the automatic DNA synthesizer. Following ordinary deprotection, DNA
was recovered, purified with high performance liquid
chromatography, and used in the following experiments.
[0107] SEQ ID No. 1
[0108]
.sup.5'HS--(CH.sub.2).sub.6--O--PO.sub.2--O-ACTGGCCGTCGTTTTACA
.sup.3'
[0109] (4) DNA Ejection and Binding to Substrate Using BJ
Printer
[0110] The ssDNA of SEQ ID No. 1 was dissolved in a TE solution (10
mM Tris-HCl (pH 8)/1 mM EDTA aqueous solution) to a final
concentration of about 400 mg/ml to prepare a ssDNA solution
(accurate concentration is calculated from absorbance).
[0111] An aqueous solution containing glycerin at 7.5 wt %, urea at
7.5 wt %, thiodiglycol at 7.5 wt %, and an acetylene alcohol
(Product name: Acetylenol EH; Kawaken Fine Chemical Co., Ltd.)
having the above general formula (I) at 1 wt % was prepared and
added to the DNA solution to adjust a final concentration of the
ssDNA to 8 .mu.M. This liquid had surface tension in a range of
30-50 dyn/cm and viscosity of 1.8 cps (E-type viscometer: Tokyo
Keiki Co., Ltd.). This liquid was filled in an ink tank of a bubble
jet printer (Product name: BJC620; Canon Inc.) and the ink tank was
mounted on a bubble jet head. The bubble jet printer used here
(Product name: BJC620; Canon Inc.) had been modified to enable
printing on a plate. This bubble jet printer can print with a
resolution of 360.times.720 dpi. The glass plate treated in the
above (2) was then mounted on this printer and the liquid
containing the probe nucleic acid was spotted on the glass plate.
The distance between the nozzle tip of the bubble jet head and the
surface of the glass plate was 1.2-1.5 mm. The conditions for
spotting were set in such a manner that the liquid was spotted once
followed by 2 idle ejections in a direction of 360 dpi and then
spotted once followed by 5 idle ejections in a direction of 720
dpi. After completion of spotting, the glass plate was left to
stand in a humid chamber for 30 minutes to complete the reaction
between the maleimido groups on the glass plate surface and the
thiol groups at the terminus of the nucleic acid probes. The amount
of the DNA solution ejected by one ejection operation of the
printer was about 24 pl.
[0112] (5) Blocking Reaction
[0113] After completion of the reaction between the maleimido group
and the thiol group, the glass plate was washed with an 1 M NaCl 50
mM phosphate buffer solution (pH 7.0) to rinse completely away the
liquid containing DNA on the surface of the glass plate. Then, the
glass plate was immersed in a 2% bovine serum albumin aqueous
solution and left for 2 hours to proceed a blocking reaction.
[0114] (6) Hybridization Reaction
[0115] A ssDNA with a base sequence complementary to DNA of SEQ ID
No. 1 was synthesized using an automatic DNA synthesizer, and
rhodamine was bound to its 5'-terminus to obtain a labeled ssDNA.
This labeled ssDNA was dissolved in 1 M NaCl/50 mM phosphate buffer
solution (pH 7.0) to a final concentration of 1 .mu.M. The probe
array subjected to the blocking treatment obtained in the above (5)
was immersed in this solution at room temperature (25.degree. C.)
for 3 hours to proceed a hybridization reaction. Then, the probe
array was washed with 1 M NaCl/50 mM phosphate buffer solution (pH
7.0) to wash away the ssDNA which had not been hybridized with the
probe nucleic acid. Then, the fluorescence intensity of each spot
of the probe array was quantified using the image analyzer (Product
name: ARGUS; Hamamatsu Photonics Co., Ltd.).
[0116] (7) Results
[0117] The fluorescence intensity of the spots of the nucleic acid
of SEQ ID No. 1 completely matched with the labeled ssDNA was
4,600. In addition, the probe array in which the respective spots
emitted fluorescence after hybridization was observed using a
fluorescent microscope (Nikon Corp.). The results indicated that,
in the probe array of this example,
[0118] (a) Each spot was almost round and had a diameter in a range
of about 70-100 .mu.m;
[0119] (b) There were spaces of about 100 .mu.m, which was almost
the same as the diameter of each spot, between adjacent spots so
that each spot was clearly independent;
[0120] (c) The columns and rows of the spots were arranged in
lines.
[0121] These facts are very effective in automatic detection, etc.
of hybridized spots on a probe array.
EXAMPLE 2
[0122] Manufacturing of Nucleic Acid Probe Array Using Bubble Jet
Printer and Detection of Target Nucleic Acid Using the Probe
Array
[0123] (1) A glass plate for a probe array was prepared in the same
manner as in (1) and (2) of Example 1.
[0124] (2) Synthesis of Probe DNA
[0125] Single-stranded nucleic acids of SEQ ID Nos. 1-4 were
synthesized using an automatic DNA synthesizer. The ss nucleic
acids of SEQ ID Nos. 2-4 were as follows: from the ss nucleic acid
of SEQ ID No. 1 used in Example 1, one base differs in SEQ ID No.
2, 3 bases in SEQ ID No. 3, and 6 bases in SEQ ID No. 4. A thiol
(--SH) group was introduced at each terminus of the ssDNAs of SEQ
ID Nos. 1-4 using Thiol-Modifier (Glen Research Co., Ltd.) during
synthesis on the automatic DNA synthesizer. Following ordinary
deprotection, DNA was then recovered, purified with high
performance liquid chromatography, and used in the following
experiments. The sequences of SEQ ID Nos. 2-4 are shown below:
1 .sup.5'HS-(CH.sub.2).sub.6-O-PO.sub.2-O-ACTGGCCGTTGTTTTACA.sup.3'
SEQ ID No. 2: .sup.5'HS-(CH.sub.2).sub.6-O-PO.sub-
.2-O-ACTGGCCGCTTTTTTACA.sup.3' SEQ ID No. 3:
.sup.5'HS-(CH.sub.2).sub.6-O-PO.sub.2-O-ACTGGCATCTTGTTTACA.sup.3'
SEQ ID No. 4:
[0126] (3) DNA Probe Ejection and Binding to Substrate Using BJ
Printer
[0127] The ssDNAs of SEQ ID Nos. 1-4 above were used to prepare 4
ejection liquids by the method similar to that described in (4) of
Example 1. The respective liquids were filled in 4 ink tanks of a
bubble jet printer used in Example 1 and the respective ink tanks
were mounted on the bubble jet heads. The glass plate prepared in
(1) was mounted on the printer, and the 4 nucleic acid probes were
spotted in respective 4 areas of 3.times.3 mm on the glass plate.
The spotting pattern in each area was the same as that in Example
1. After completion of spotting, the glass plate was left in a
humidified chamber for 30 minutes to react the maleimido group and
the thiol group.
[0128] (4) Blocking Reaction
[0129] After completion of the reaction between the maleimido group
and the thiol group, the glass plate was washed with a 1 M NaCl/50
mM phosphate buffer solution (pH 7.0) to rinse completely away the
solution containing DNA on the surface of the glass plate.
[0130] Then, the glass plate was immersed in a 2% bovine serum
albumin aqueous solution and left for 2 hours to proceed a blocking
reaction.
[0131] (5) Hybridization Reaction
[0132] A ssDNA with a base sequence complementary to DNA of SEQ ID
No. 1 was synthesized using an automatic DNA synthesizer, and
rhodamine was bound to its 5'-terminus to obtain a labeled ssDNA.
This labeled ssDNA was dissolved in an 1 M NaCl/50 mM phosphate
buffer solution (pH 7.0) to a final concentration of 1 .mu.M. The
probe array obtained in (4) was subjected to a hybridization
reaction for 3 hours. Then, the probe array was washed with 1 M
NaCl/50 mM phosphate buffer solution (pH 7.0) to wash away the
ssDNA which had not been hybridized with the probe nucleic acid.
Then, the respective spots of the probe array were observed using a
fluorescent microscope (Nikon Corp.) and the amounts of
fluorescence were quantified using the image analyzer (Product
name: ARGUS; Hamamatsu Photonics Co., Ltd.).
[0133] (6) Results
[0134] The fluorescence intensity of the spots of the DNA probe of
SEQ ID No. 1 completely matched with the labeled ssDNA was 4,600,
while the fluorescence intensity was 2,800 for the spots of the DNA
probe of SEQ ID No. 2 containing one mismatched base. For the spots
of the DNA probe of SEQ ID No. 3 having 3 mismatched bases, the
fluorescence intensity was 2,100, which was less than half that for
the completely matched probe. No fluorescence was observed for DNA
of SEQ ID No. 4 containing 6 mismatched bases. The above result
indicates that a completely complementary ssDNA was specifically
detected on the DNA array substrate.
EXAMPLE 3
[0135] Concentration of DNA Probe Solution and Bubble Jet Ejection
Properties
[0136] (1) Synthesis of DNA Probe
[0137] A ssDNA of SEQ ID No. 5 shown below was synthesized using an
automatic DNA synthesizer and dissolved in a TE solution (10 mM
Tris-HCl (pH 8)/1 mM EDTA aqueous solution) to concentrations of
about 0.2 mg/ml, 2 mg/ml, and 1.5 mg/ml to prepare DNA probe
solutions of 3 different concentrations (accurate concentrations
were calculated from absorbance).
[0138] SEQ ID No. 5:
[0139] .sup.5' GCCTGATCAGGC.sup.3'
[0140] (2) Ejection by BJ Printer
[0141] An aqueous solution containing glycerin at 7.5 wt %, urea at
7.5 wt %, thiodiglycol at 7.5 wt %, and acetylene alcohol (Product
name: Acetylenol EH; Kawaken Fine Chemical Co., Ltd.) having the
above general formula (I) at 1 wt % was prepared, added to the 0.2
mg/ml probe solution prepared in (1), and adjusted a final
concentration to about 0.02 mg/ml (3 .mu.M). This solution was
filled in an ink tank of a bubble jet printer used in Example 1 and
the ink tank was mounted on a bubble jet head used in Example
1.
[0142] An aluminum plate of A4 size was mounted on the printer and
the liquid was spotted to an area of 3.times.5 square inch of the
aluminum plate. The condition of spotting was set so as to perform
spotting in a density of 360.times.720 dpi in the above area. A
commercial ink for BJ620 was first printed on the aluminum plate as
a control. This operation was performed on a total of 4 aluminum
plates.
[0143] The nucleic acid probe spotted on the respective aluminum
plates was recovered using the TE solution and purified by a gel
filtration method. The amounts of the recovered nucleic acid probe
purified were measured by absorbance. The recovery of the nucleic
acid probe theoretically obtained is as follows. That is, a volume
of a droplet ejected from the head of the printer used in this
example was 24 picoliters. Then, since there were 4 aluminum plates
on which the solution was spotted in an area of 3.times.5 square
inch at a density of 360.times.720 dpi, the following equation was
obtained:
24 (picoliters).times.(720.times.360).times.(3.times.5).times.4
plates=373 .mu.l
[0144] Absorbance at 260 nm of the probe nucleic acid for this
volume and absorbance at 260 nm of recovered nucleic acid probe are
shown in FIG. 3.
[0145] (3) The operation identical to that described in (2) was
performed on the probe solutions at concentrations of 2 mg/ml and
15 mg/ml. The final concentrations of the nucleic acid probe of the
respective ejection liquids were 30 .mu.M (0.2 mg/ml) and 225 .mu.M
(1.5 mg/ml). Absorbance of the probe nucleic acid recovered from
the respective solutions and absorbance of the probe nucleic acid
in amounts theoretically obtained are shown in FIG. 3.
[0146] (4) Results
[0147] As shown in FIG. 3, the amounts of a nucleic acid probe
actually ejected were close to the values theoretically
anticipated. From this, in ejection of a nucleic acid probe using
the bubble jet method, no quantitative loss of the nucleic acid
probe due to burning and sticking of the nucleic acid probe to the
heater of the bubble jet head was observed. No troubles in the
head, such as no ejection, occurred during the step of spotting on
the aluminum plate using liquids of various concentrations. A
macroscopic comparison with the spots of the ink for a bubble jet
printer spotted on the aluminum plate as a control and the spots of
the nucleic acid probe showed that the spotting status for the
spots formed using the liquids at concentrations of 3 .mu.M and 30
.mu.M was similar to that for the ink spot. The spots formed using
the liquid at a concentration of 225 .mu.M exhibited some disorders
as compared with the ink spot.
EXAMPLE 4
[0148] Influence of Bubble Jet Process on Nucleic Acid Probe
[0149] (1) Synthesis of Nucleic Acid Probe
[0150] A nucleic acid probe comprised of 10 mer adenylic acids
(abbreviated as "A" hereinafter) (synthetic substance), oligoA
(40-60 mer; Pharmacia Co., Ltd.), and poly(dA) (300-400 mer;
Pharmacia Co., Ltd.) were respectively diluted with a TE solution
to prepare solutions of the nucleic acid probes of different base
lengths at a final concentration of 1 mg/ml. The base sequence of
the 10-mer probe (SEQ ID No. 6) is shown below:
Sequence CWU 1
1
14 1 18 DNA Artificial Sequence modified_base 1 Thiol group bound
at the 5'-terminus 1 acattttgct gccggtca 18 2 18 DNA Artificial
Sequence modified_base 1 Thiol group bound at the 5'-terminus 2
acattttgtt gccggtca 18 3 18 DNA Artificial Sequence Synthetic 3
actggccgct tttttaca 18 4 18 DNA Artificial Sequence Synthetic 4
actggcatct tgtttaca 18 5 12 DNA Artificial Sequence Synthetic 5
gcctgatcag gc 12 6 10 DNA Artificial Sequence Synthetic 6
aaaaaaaaaa 10 7 18 DNA Artificial Sequence modified_base 1 Cysteine
residue bound at the N'-terminus 7 actggccgtc gttttaca 18 8 18 DNA
Artificial Sequence modified_base 1 Cysteine residue bound at the
N'-terminus 8 actggccgtt gttttaca 18 9 18 DNA Artificial Sequence
modified_base 1 Amino group bound at the 5'-terminus 9 tgaccggcag
caaaatgt 18 10 18 DNA Artificial Sequence modified_base 1 Amino
group bound at the 5'-terminus 10 tgaccggcac caaaatgt 18 11 18 DNA
Artificial Sequence modified_base 1 Amino group bound at the
5'-terminus 11 tgacccgcac caatatgt 18 12 18 DNA Artificial Sequence
modified_base 1 Thiol group bound at the 5'-terminus 12 tgacccgcag
caaaatgt 18 13 18 DNA Artificial Sequence modified_base 1 Thiol
group bound at the 5'-terminus 13 tgaccggcac caaaatgt 18 14 18 DNA
Artificial Sequence modified_base 1 Thiol group bound at the
5'-terminus 14 tgacccgcac caatatgt 18
* * * * *