U.S. patent application number 11/118357 was filed with the patent office on 2005-09-01 for reactive probe chip, composite substrate and method for fabrication of the same.
This patent application is currently assigned to Ebara Corporation. Invention is credited to Fukunaga, Akira, Hirose, Masayoshi, Nagasawa, Hiroshi.
Application Number | 20050191699 11/118357 |
Document ID | / |
Family ID | 27342886 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191699 |
Kind Code |
A1 |
Nagasawa, Hiroshi ; et
al. |
September 1, 2005 |
Reactive probe chip, composite substrate and method for fabrication
of the same
Abstract
A reaction probe chip which is prepared by loading a reactive
probe on fine pieces of carrier such as particles, tile-like plates
and then arraying and immobilizing the reactive probe-loaded
carrier on a base material. The carrier fine pieces such as
particles, tile-like plates and the like are porous or have a
reactive surface, and the base material is preferably a thin
inorganic plate or a thin organic plate is disclosed. The inorganic
base material is preferably a glass slide or silicon wafer, and the
organic base material is preferably a polyester film or
polyethylene film. In case the porous carrier pieces are used, the
reactivity of the inner surfaces of the porous carrier pores should
be maintained during array or immobilization process of the
reactive probe-loaded carrier. A composite substrate characterized
in that on at least a section of the surface thereof, a plurality
of porous regions are orderly arranged as compartments by
non-porous regions, or a plurality of non-porous regions are
orderly arranged as compartments by porous regions is also
disclosed. The porous solid is preferably porous glass or porous
ceramic, the porous glass is preferably split-phase porous glass,
and the surface is preferably flattened by a process such as
polishing.
Inventors: |
Nagasawa, Hiroshi; (Osaka,
JP) ; Fukunaga, Akira; (Kanagawa-ken, JP) ;
Hirose, Masayoshi; (Kanagawa-ken, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Ebara Corporation
Tokyo
JP
|
Family ID: |
27342886 |
Appl. No.: |
11/118357 |
Filed: |
May 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11118357 |
May 2, 2005 |
|
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|
09820778 |
Mar 30, 2001 |
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6897021 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00626
20130101; B01J 2219/00527 20130101; G01N 33/54346 20130101; B01J
2219/00612 20130101; B01J 2219/0063 20130101; B01J 2219/00659
20130101; B01J 2219/00497 20130101; B01J 2219/00585 20130101; B01J
2219/00644 20130101; B01J 2219/00596 20130101; G01N 33/545
20130101; B01J 2219/0061 20130101; C40B 40/06 20130101; B01J
2219/00641 20130101; G01N 33/552 20130101; B01J 2219/00317
20130101; B01J 2219/00621 20130101; B01J 2219/00605 20130101; B01J
2219/00637 20130101; B01J 2219/00617 20130101; B01J 2219/00608
20130101; C40B 60/14 20130101; B01J 2219/00722 20130101; B01J
2219/0059 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2000 |
JP |
94529/2000 |
Sep 27, 2000 |
JP |
294462/2000 |
Sep 27, 2000 |
JP |
294463/2000 |
Claims
What is claimed is:
1. A reactive probe chip for detecting target functional molecules,
comprising: one or a plurality of first carrier probes, wherein
said first carrier probe is a porous carrier in the form of a
particle, having immobilized within the pores thereof a first
reactive substance capable of bonding a first target molecule; one
or a plurality of second carrier probes, wherein said second
carrier probe is a porous carrier in the form of a particle, having
immobilized within the pores thereof a second reactive substance
capable of bonding a second target molecule; and a substrate
material, wherein said one or a plurality of first carrier probes
and said one or a plurality of second carrier probes are
immobilized on a surface of said substrate material.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a reactive probe chip
capable of recognizing a plurality of functional molecules, to be
used for gene diagnosis, physiological function diagnosis and the
like, and to a method for its fabrication.
[0002] Also, the present invention relates to a composite substrate
wherein on at least a section of the surface thereof, a plurality
of porous regions are arrayed as compartments encompassed by
non-porous regions, or a plurality of non-porous regions are
arrayed as compartments encompassed by porous regions, and to a
method for its fabrication.
[0003] Detection of polymorphisms due to gene mutations, and
particularly single base (codon) mutations, is not only effective
for diagnosis of cancer and other diseases resulting from
mutations, but is also necessary for indication of drug
responsiveness and side-effects, and can be helpful for the
analysis of the causative genes of multiple factor diseases, and
for predictive medicine. The use of "DNA chips" for detection is
known to be effective. The hitherto utilized "Gene Chip" by
Affymetrix, which is a DNA chip containing immobilized short DNA
chains, usually comprises over 10,000 oligo DNA fragments (DNA
probes) mounted on an approximately 1 cm square silicon or glass
plate using a photolithographic technique.
[0004] When a fluorescently-labeled DNA sample to be examined is
allowed to flow over the DNA chip, the DNA fragments having
sequences complementary to the probes of the DNA chip bind to the
probes to perform detection of only those sections by their
fluorescence, and thereby detecting and quantifying the specific
sequences of DNA fragments in the DNA sample. It has already been
demonstrated that this method can detect cancer gene mutations and
gene polymorphisms.
[0005] Microarrays with cDNA immobilized on slide glass may also be
used.
[0006] On the other hand, porous solids have been widely used in
the prior art as carriers for catalysts, enzymes, microorganisms
and the like, and are utilized as sites for various reactions. They
are also used as functional materials for adsorption and
separation, or as materials providing low heat or electrical
conductivity. Porous glass or porous ceramics are used as porous
solids, and methods of controlling the pore sizes or imparting
functional groups have provided properties suitable for specific
uses.
[0007] There were three principal problems in the prior art. DNA
chips employing photolithography require at minimum four photomasks
for each step of synthesis, and the photolithography, coupling and
washing must be repeated four times. Since this is repeated the
required times depending on the chain length, the cost is high
(problem 1). Also, it is necessary to change each photomask in
order to change the pattern, for which reason, DNA chips with
various designs could not be flexibly fabricated (problem 2).
[0008] That is, although the types of DNA probes to be immobilized
on the DNA chip are previously decided and although it is easy to
fabricate and supply such DNA chips at locations where the
necessary equipment is available, different photomasks must be
prepared for synthesis of each base in the probe. Therefore, the
reaction process has may steps and it is difficult to fabricate in
a flexible manner, DNA chips containing DNA probes for different
purposes. High costs are also incurred. When the number of DNA
probes required is small, the degree of integration of the DNA
probes on the chip need not be so high. Rather, it is sometimes
desirable for the chip to have the desired DNA probes immobilized
in a more convenient manner. The DNA chip must also be provided at
low cost and with high stability if it is clinically used to detect
DNA polymorphisms of individuals.
[0009] Alternatively DNA Microarray chips prepared by spotting a
solution of synthesized oligonucleotide at high density, is
proposed. In this process, modifying groups are introduced after
synthesis of the oligonucleotides and then the modified
oligonucleotides are cut out and released from the carrier and
purified. Further the purified oligonucleotides are reacted with
the functional groups previously introduced onto the substrate
glass. Thus, the process is very complex and the cost is therefore
high (problem 3) as of the DNA chips produced by
photolithography.
[0010] With respect to substrates for probe chips, porous solids
have a wide variety of applications, for example, carrier of probe
chips such as DNA chips, and the like, but the use of porous solids
having continuous porous regions throughout the entire surface has
been limited in that only one function can be loaded on such a
single and uniform substrate.
[0011] However, in recent advancements in scientific techniques,
downsized devices is in ever greater demand and it has been desired
to develop a composite substrate having unique utilities not found
in the prior art, and a substrate having different reaction sites
with a plurality of functions on the single substrate whereby the
local areas of the substrate can be thermally or electrically
insulated each other.
[0012] In light of the problems in the prior art, it is an object
of the present invention to provide a composite substrate as a
single substrate allowing a plurality of different functions to be
loaded, as well as a method for its fabrication.
SUMMARY OF THE INVENTION
[0013] The present invention solves the aforementioned problems
with respect to reactive probe chips and to composite substrates by
way of the following means.
[0014] According to one aspect of the invention, porous carrier
particles are used as a carrier fine pieces to be loaded with
reactive substances or reactive probes.
[0015] (1) A reactive detecting chip characterized in that fine
porous carrier particles having reactive substances, which have
ability to bond different detection targets and which are loaded on
the inner surfaces of the porous particle pores, serve as a whole
porous carrier particle probes. They are arrayed and bound or
immobilized on at least one of a plurality of microcompartments
provided on a base material, while maintaining the reactivity of
the inner surfaces of the porous carrier particle pores.
[0016] (2) A reactive probe chip according to (1) above, wherein
the porous carrier particles loaded with the reactive probes are of
a material having a bonding surface, such as porous glass, silica
gel or ion-exchange resin.
[0017] (3) A reactive probe chip according to (1) or (2) above,
wherein the pore size of the porous carrier particles ranges from
10 nm to 1 .mu.m, and the particle size is from 1 .mu.m to 100
.mu.m.
[0018] (4) A reactive probe chip according to any one of (1) to (3)
above, wherein the base material immobilizing the porous carrier
particles loaded with the reactive is an inorganic base material or
organic base material.
[0019] (5) A reactive probe chip according to any one of (1) to (4)
above, wherein the reactive probes to be loaded in the porous
carrier particles are DNA, RNA or PNA (peptide nucleic acid) or
fragments thereof, oligonucleotides of any desired base sequence,
antigens, antibodies or epitopes, or enzymes, proteins or their
partial polypeptide chains having the target functions.
[0020] (6) A method for fabrication of a loaded porous carrier
particles wherein a solid phase method is used to synthesize
oligonucleotides having desired base sequences or proteins having
desired structures on porous carrier particles, and they are used
as they are.
[0021] (7) A method for fabrication of a loaded porous carrier
particles, which comprises binding DNA, RNA or PNA (peptide nucleic
acid) or fragments thereof, oligonucleotides of any desired base
sequences, antigens, antibodies or epitopes, or enzymes, proteins
or polypeptide chains which are a part of the proteins and have the
functions, to porous carrier particles using a binding
material.
[0022] (8) A method for fabrication of a reactive probe chip,
characterized in that the one or more loaded porous carrier fine
particles fabricated according to (5) or (6) above are arrayed and
bound or immobilized in at least one of a plurality of
microcompartments provided on a base material while maintaining the
reactivity of the inner surfaces of the porous carrier particle
pores.
[0023] According to one aspect of the invention, oligonucleotides
having any desired base sequence or proteins having any desired
structure may be synthesized on porous carrier particles with a
surface of the binding ability, such as porous glass, silica gel or
ion-exchange resin, by a solid phase method. Alternatively, the
reactive substances or probes such as DNA, RNA or PNA (peptide
nucleic acid) or fragments thereof, oligonucleotides of any desired
base sequences, antigens, antibodies or epitopes, or enzymes,
proteins or their fragments staining their functions, are bound to
the porous carrier particles using some sort of binding material.
Thus, the loaded porous carrier particle probes are produced.
[0024] The produced particulate probes may be used alone or in
combination, and bound or immobilized utilizing a dispenser or
printing method, on at least one of a plurality of
microcompartments provided on a base material. The base material is
selected from an inorganic base material such as a slide glass or
silicon wafer or an organic base material such as a polyester film
or polyethylene film. The probes are bound on the base material
while maintaining the reactivity of the inner surfaces of the
porous carrier particle pores.
[0025] For the binding and immobilization of the porous carrier
particles in an orderly fashion on the base material according to
the invention, only the outer surfaces of the carrier particles are
used for immobilization, and a protective measure such as
impregnation with water is utilized in order to accomplish
immobilization without incurring damage to the inner pore surfaces
by an adhesive component used for the immobilization.
[0026] In accordance with another aspect of the invention, the
aforementioned problems can be solved by arraying and binding
reactive substances or probes on tile-like carriers, and then
arraying and immobilizing each tile-like carrier on a base
material.
[0027] Another aspect of the present invention solves the
aforementioned problems by way of the following means.
[0028] (1) A reactive probe chip characterized in that tile-like
carriers loaded with reactive substances are arrayed and
immobilized on a base material.
[0029] (2) A reactive probe chip according to (1) above, wherein
the tile-like carriers loaded with the reactive substances are of a
material having a reactive surface, and then the base material
immobilizing the carriers includes a thin inorganic or organic
plate.
[0030] (3) A method for fabrication of a reactive probe chip,
characterized in that enzymes, antigens, DNA fragments, antibodies,
epitopes or proteins are arrayed and immobilized on tile-like
carriers, and each of the loaded carriers is immobilized in an
orderly fashion in separate compartments on the base material.
[0031] (4) A method for fabrication of a reactive probe chip
according to (3) above, wherein after synthesizing oligonucleotides
with desired base sequences on tile-like carriers, each carrier is
immobilized in an orderly fashion into separate compartments on the
base material.
[0032] (5) A method for fabrication of a reactive probe chip
according to (3) or (4) above, wherein the tile-like carriers are
each a plate with a square shape having a size of from 50 .mu.m to
5 mm on each side, or a hexagonal or circular shape, and they are
mechanically attached and immobilized on the base material.
[0033] In still another aspect of the present invention, an
excellent composite substrate that can exhibit a plurality of
different functions as a single substrate, is provided.
[0034] The aspect of the present invention has been completed by
focusing attention on applicability not found in the prior art and
by providing a composite substrate wherein either a plurality of
porous regions are arrayed and comparted by non-porous regions, or
a plurality of non-porous regions are arrayed and comparted by
porous regions on at least a section of the surface of a porous
solid conventionally used in the form of particles. The composite
substrate provides reaction sites with a plurality of different
functions on the same substrate, or to thermally or electrically
insulate specific regions of the substrate.
[0035] Still another aspect of the present invention solves the
aforementioned problems by way of the following means.
[0036] (1) A composite substrate characterized in that on at least
a section of the surface thereof, a plurality of porous regions are
arrayed on a substrate material and comparted by non-porous
regions, or a plurality of non-porous regions are arrayed and
comparted by porous regions.
[0037] (2) A composite substrate according to (1) above, wherein a
composite substrate comprising both porous regions and non-porous
regions has a surface flattened by, for example, a polishing
process.
[0038] (3) A method for fabrication of a composite substrate,
wherein the composite substrate according to (1) above is produced
by situating a separately formed porous solid at predetermined
regions on a non-porous substrate.
[0039] (4) A method for fabrication of a composite substrate
according to (3) above, characterized in that the formation of the
composite substrate is accomplished by situating a plurality of
porous solid precursors at predetermined regions on a non-porous
substrate and producing pores in the porous solid precursors placed
on the substrate.
[0040] (5) A method for fabrication of a composite substrate
according to (3) above, wherein formation of the plurality of
porous regions is accomplished by producing pores in a plurality of
predetermined porous solid precursor regions on a substrate the
entire surface of which is a porous solid precursor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is an illustration showing a process for fabrication
of a reactive probe chip using a carrier particle immobilizing
apparatus according to Example 1.
[0042] FIG. 2 is an illustration showing a process for fabrication
of a reactive probe chip using a carrier particle immobilizing pin
according to Example 2.
[0043] FIG. 3 is an illustration showing a process for fabrication
of a reactive probe chip using a carrier particle immobilizing pin
according to Example 3.
[0044] FIG. 4 is an illustration showing a process for fabrication
of a reactive probe chip by screen printing according to Example
4.
[0045] FIG. 5 is an illustration of a fabrication process for a
reactive probe chip according to the invention.
[0046] FIG. 6 is an illustration of a fabrication process for an
alumina substrate having a plurality of porous alumina bead regions
on the surface.
[0047] FIG. 7 is an illustration of a fabrication process for a
glass plate having a plurality of porous glass regions on the
surface.
[0048] FIG. 8 is an illustration of a process for fabrication of a
glass plate having a plurality of porous glass regions by coating
of a porous glass precursor on the surface.
[0049] FIG. 9 is an illustration of a process for fabrication of a
quartz plate having a plurality of porous glass regions by a
split-phase method with coating of borosilicate glass particles on
the surface.
DETAILED DESCRIPTION OF THE INVENTION
[0050] It is a characteristic of one aspect of the present
invention that the oligonucleotides constituting the DNA probes are
bound on the inner pore surfaces of the porous carrier particles,
so that the porous carrier particles act as "loaded porous carrier
particulate probes" as they are.
[0051] Another aspect of the present invention is directed to a
reactive probe chip characterized in that a plurality of
particulate or tile-like carriers loaded with reactive probes are
immobilized in separate compartments on the surface of a base
material. Since a plurality of reactions can be conducted
simultaneously, many types of reactive probes are used. The
compartments for placing the tile-like carriers are preferably
arranged in such a manner that the plurality of particulate or
tile-like carriers loaded with the reactive probes can be arrayed
by a mechanical or automatic procedure. The compartments are
preferably formed in well-ordered array.
[0052] It further relates to a method for fabrication of a reactive
probe chip, characterized in that after synthesizing
oligonucleotides having desired base sequences on tile-like
carriers, each tile-like carrier is immobilized in separate
compartments on a base material. In this case, it is necessary to
use means that can rapidly and sequentially convene the small
tile-like carriers loaded with the oligonucleotides from contacting
the surface in the compartments on the base material.
[0053] A composite substrate of still another aspect of the
invention has a plurality of porous regions on at least a section
of its surface, which regions are surrounded and comparted by
non-porous regions.
[0054] Alternatively, the composite substrate has a plurality of
non-porous regions that are surrounded and comparted by porous
regions.
[0055] In the former case, for example, a specific catalytic
reaction or enzyme reaction can take place only in the
compartmented regions. Thus, if a sample is exposed to a substrate
having a plurality of such regions comparted isolatedly each other,
the sample will be allowed to simultaneous testing of the presence
of reactivity for the plurality of reactions.
[0056] In the latter case, for example, a semiconductor fabrication
process may be employed to partition a plurality of wirings
(non-porous regions) by insulative porous regions and to form a
highly insulated electrical wiring on the substrate.
[0057] The reactive substances or probes carried on the porous
particles may be any of those which react with detection targets,
for example, oligonucleotides, enzymes, antigens, antibodies,
epitopes or proteins. A method wherein the reactive probes
immobilized on each of the carriers are arrayed and set in the
compartments on the base material surface can stably and flexibly
produce the chips.
[0058] Two methods exist for loading the reactive substances on the
inner pore surfaces of the porous carrier particles. One method is
that oligonucleotides having desired base sequences or proteins
with desired structures are synthesized on the porous carrier
particles by a solid-phase method, to prepare the reactive
substances in situ.
[0059] The other method is as follows. A purified extract from
animal or plant cells or a synthesized reactive substance is bound
by some method, for example, in the case of porous glass, reacting
amino silane with the porous glass surface to bind the amino
groups. Using glutaraldehyde, various enzymes can be bond to the
formed amino groups.
[0060] The reaction between the detection target and the reactive
substances occurs in the pores of the porous carrier particles, and
since the detection target must be incorporated in the pores of the
porous carrier particles, the pores of the porous carrier particles
must be large enough that the detection target can be moved into by
the incorporated diffusion. The pore size usually ranges from 10 nm
to 1 .mu.m, and preferably 50 nm to 200 nm.
[0061] Reaction in the pores of the porous carrier particles can
prevent side-reaction with contaminants, and the larger reactive
surface area allows more accurate detection.
[0062] According to one aspect of the invention, highly stable,
reactive substance-loaded porous carrier particles or tile-like
plates can be stored because the reactive substance synthesized on
the carrier is used as it is, or because the substances immobilized
on the carriers in used in the detection.
[0063] While there are no particular restrictions on the method of
arraying and immobilizing the reactive substance on the base
material, a protective solution, such as a single amount of water
may be contained in the carrier particles to protect the inner
surface of the pores and then an inorganic base material such as
silica sol is added to the carrier particles to make a slurry,
which is arrayed on a support by use of a dispenser. This method is
useful to fabricate a small number of reactive probe chips, but in
order to mass-produce the reactive probe chips, the slurry may be
used in the form of printing ink to form the array pattern by a
multi-color printing method.
[0064] When using such methods, it will be difficult to form a
slurry or ink if the size of the "loaded porous carrier particles"
is very large, and therefore the porous carrier particle size
preferably ranges from 1 .mu.m to 100 .mu.m, and more preferably
from 3 .mu.m to 20 .mu.m. This is because a large particle size is
preferred from the standpoint of handleability during the process
of loading the reactive substance, but a small particle size is
preferred when the porous carrier particles are immobilized after
the reactive substances have been loaded; nevertheless, large
grains may be used so long as they can be arrayed.
[0065] By using these methods it is possible to achieve stable and
flexible production. The base material may be of a material that is
stable and is not deteriorated upon using it in the detection
system, but it must have surface properties suitable for
immobilizing the porous carrier particles. Glass plates such as
quartz glass, borosilicate glass and the like, or inorganic base
materials such as silicon wafers are preferred.
[0066] Modification of the method of binding the porous carrier
particles will enable to use of an organic base material such as a
polyester film or polyethylene film, and in some cases even paper
materials can be used. The base material surface may be
appropriately treated for the purpose of adjusting the affinity
with the carrier binding material.
[0067] The porous carrier particles must be of a material that can
carry, as the reactive substances, any proteins having the desired
structures or the oligonucleotides having the desired base
sequences. The porous materials having a binding ability, such as
porous glass, silica gel, ion-exchange resin and the like are
preferred, among which porous glass is most preferred because its
surface reactivity associated with pore sizes can be
controlled.
[0068] The surface of the porous carrier particle is preferably
subjected to appropriate surface treatment in order to adjust its
affinity with the reactive substance or reactive probe.
[0069] There are no particular restrictions on the shape of the
base material, and for example, it may be thin plate such as a film
or sheet, or in a cubical, rod-like, cord-like or spherical
shape.
[0070] When a thin plate is used, there are no particular
restrictions on the thickness or size of the base material, and the
thickness of the base material may be easily determined in
consideration of the form stability required for the base material.
The size of the base material may be easily determined on
consideration of the number of microcompartments to be formed in
the base material surface.
[0071] The microcompartments on the base material surface according
to the invention are imaginary compartments, which are defined by
imaginarily formed partitions and not materially separated.
[0072] The "reactivity" of the "reactive substances or reactive
probes" according to the invention refers to not only a change in
their chemical structure by ionic bonding or covalent bonding
through a chemical reaction, but also the property capable of
forming binding states with other substances due to Van der Waals
forces, hydrogen bonding, coordination bonding, chemical
adsorption, physical adsorption or the like.
[0073] Such reactive substances, which are alternatively referred
to as reactive probes, include proteins with any desired structures
and oligonucleotides with any desired base sequences, and naturally
there are no restrictions on these.
[0074] There are no particular restrictions on the degree of
integration of the microcompartments, i.e., the compartments for
the reactive substances, in a reactive probe chip according to the
invention. Since the degree of integration required or convenient
will differ depending on the use of the reactive probe chip, the
degree of integration may be appropriately changed to conform to
the use.
[0075] As an example, there may be 100 or more micro-compartments
per cm.sup.2 of the reactive probe chip surface, and if the base
material and reactive substances are appropriately selected, about
10,000 microcompartments per cm.sup.2 of surface can be formed.
[0076] Since the reactive probe chip of the invention has the
reactive substances loaded on porous carrier particles, tiles or
the like, the substance are not easily penetrated and released on
the base material. Also, since the size of porous carrier particles
is small, it is possible to immobilize a solution containing the
particles at high density in the compartments on the base
material.
[0077] The reactive substances carried on the porous carrier solid
may be the same types of substances or different types of
substances, depending on the use of the reactive probe chip. From
the viewpoint of working efficiency, it is preferred to load a
plurality of reactive substances at one time, and it is more
preferred to load all of the reactive substances at one time.
[0078] The reactive substance-loaded porous carrier solid may be
separately prepared and stored, or if necessary, they may be
immobilized on the base material in the necessary combinations.
Especially, it is practical in the case of
oligonucleotide-synthesized porous carrier particles or tiles,
because a usual synthesis process can be used.
[0079] The size and shape of the carriers may be selected as
desired, but considering immobilization of the carriers loaded with
a plurality of different reactive probes onto the substrate, when
tile-like carrier is used, it is preferably a plate with a square
shape having a size of from 50 .mu.m to 5 mm on each side, or a
hexagonal or circular shape, and squares of 100 .mu.m to 1 mm are
particularly preferred. The thickness will depend on the size, but
it is preferably in the range of 100-200 .mu.m. The immobilization
of the tile-like carriers on the base material may be mechanically
attached and immobilized on the substrate using an adhesive that
does not affect the reaction.
[0080] For example, an acrylic resin may be used as an adhesive for
immobilization of the tile-like carriers onto the substrate.
[0081] The apparatus used for immobilization of the tile-like
carriers onto the substrate may be one used for processing and
conveyance of micromembers used in the production of semiconductor
devices.
[0082] In case the tile-like carrier is used, a process for
fabrication of a reaction chip according to the invention will now
be explained with reference to the attached drawings.
[0083] FIG. 5 is an illustration of the fabrication process, and in
step (a), an adhesive 2 is coated on the area in which the reactive
probes are to be set on the glass slide 1 serving as the base
material for the reaction chip.
[0084] In step (b), an array dispenser 4 with a suction chuck 5 at
the lower end is moved to Region A which contains base materials
bearing a plurality of the same reactive probe-loaded tiles 3
manufactured in advance, and a reactive probe-loaded tile 3 is
suctioned with the suction chuck 5, and then in step (c), the array
dispenser 4 is moved over the glass slide 1, disengaging the
suction for ejection at the prescribed location, and immobilizing
the reactive probe-loaded tile 3 it has carried onto the prescribed
location.
[0085] Then, in step (d), a tile 3 is conveyed by the array
dispenser 4 from Region B which contains substrates bearing a
different plurality of reactive probe-loaded tiles 3 and is
immobilized at the subsequent prescribed location, and this
operation is repeated in order to arrange different types of
reactive probe-loaded tiles 3.
[0086] This yields, in step (e), a reaction chip 6 with different
types of reactive probe-loaded tiles 3 orderly arranged at
prescribed locations.
[0087] The porous solid for producing a composite substrate of the
still another aspect of the invention is preferably porous glass or
porous ceramic that enables to easily control the pore size and to
easily attach functional groups thereto. Split-phase porous glass
with a high density of surface hydroxyl groups, which readily
undergoes chemical modification, is especially suitable for use as
a reaction site. The sample can also be rendered homogeneous by
flattening the surface by a process such as polishing.
[0088] The split-phase porous glass has properties that allow easy
control of the pore size by appropriately selecting the heat
treatment time and temperature, and borosilicate glass is preferred
as the parent glass composition.
[0089] Porous glass compositions include
Na.sub.2O--B.sub.2O.sub.3--SiO.su- b.2 based glass with SiO.sub.2
in a range of 55-80 wt % or SiO.sub.2 in a range of 35-55 wt %, as
well as SiO.sub.2--B.sub.2O.sub.3--CaO--Al.sub.2O- .sub.3 based
glass, SiO.sub.2--P.sub.2O.sub.5--Na.sub.2O based glass,
SiO.sub.2--B.sub.2O.sub.3--CaO--MgO--Al.sub.2O.sub.3--TiO.sub.2
based glass, SiO.sub.2--B.sub.2O.sub.3--Na.sub.2O--GeO.sub.2 based
glass, SiO.sub.2--ZrO.sub.2 based glass and
GeO.sub.2--ZrO.sub.2--ThO.sub.2 based glass.
[0090] As porous ceramics there may be mentioned alumina, magnesia
and the like.
[0091] Several differing methods may be used as fabrication methods
for the composite substrate of the invention.
[0092] One is a method whereby a separately formed porous solid is
situated on prescribed regions of a non-porous substrate. The
porous solid used here may be particulate, or crushed or cut
fragments of a porous solid plate. The porous solid may also be
given a necessary function, for example, by loading a reactive
probe or reactive substance on the porous solid, prior to being
arrayed on the substrate.
[0093] For arraying on the substrate, it is preferred to use an
adhesive that does not affect the function of the reactive probes
or the porous solid, such as water glass. Pits may also be
preformed in the base material and the porous solid embedded
therein, and then the entire substrate subjected to polishing to
create a flat composite substrate.
[0094] Another method is one whereby a plurality of porous solid
precursors (substances) are situated on prescribed regions of a
non-porous substrate and pores are produced therein on the base
material. The method of situating the precursors may be a method of
attachment of solids with an adhesive or the like, or a method of
dropping a liquid or slurry and heat treating it to form a
precursor (for example a precursor substance layer) attached to the
substrate.
[0095] In this case as well, pits may be preformed at prescribed
locations in the substrate and the porous solid precursors embedded
therein, and polishing carried out after production of the pores to
obtain a flat composite substrate.
[0096] Another possible method involves preparing a substrate
composed of a porous solid precursor, or forming a porous solid
precursor layer or thin-film over the entirety of a non-porous
substrate, and producing pores in prescribed regions thereof.
[0097] The method of forming the precursor on the non-porous
substrate may be a method of attachment of a plate with an
adhesive, uniform coating of a solution or slurry by pin coating or
the like, or attachment on the substrate by heat treatment or the
like. For production of the pores, a resist may be used to cover
the other sections in order to produce pores only in the prescribed
locations. In this case, the surface of the substrate is relatively
flat prior to pore production, but if necessary it may be further
flattened by polishing.
[0098] By preparing a substrate which is porous over the entirety
and eliminating the pores in prescribed regions of the surface by
sealing treatment, with the prescribed regions selected at
locations that form the borders for the porous sections so that
they are separated by a plurality of compartments, it is possible
to form a surface with a plurality of porous regions compartmented
by non-porous formed regions.
[0099] The sealing method used to accomplish this may be a method
of irradiation of the prescribed regions with a high energy beam
such as a laser beam, or a method of chemical treatment after
coating the regions to be left porous with a resist. Laser
processing is preferred because only the regions exposed to the
laser are melted, and there is less effect on the other locations.
Laser processing can also easily form a plurality of compartmented
regions by simply shifting the laser at spacings on the substrate
to draw horizontal and vertical lines.
[0100] The present invention will be illustrated in detail by way
of examples. It is to be understood, however, that the invention is
in no way limited by these examples.
EXAMPLE 1
[0101] Proteins with different structures were synthesized on
particles of a surface-aminated ion-exchange resin powder having a
mean particle size of 10 .mu.m and an average pore size of 10
nm.
[0102] The protein-loaded porous ion-exchange resin powder was
dispersed in purified water and silica sol was added to prepare a
slurry. The slurry was then loaded in each of 1 mm-square
compartments of the surface of a glass plate 4 composed of a
borosilicate glass slide (approximately 15 cm.times.2 cm), using
the ultrathin capillary of the carrier particle binding apparatus
(dispenser) 3 shown in FIG. 1. A reactive probe chip capable of
carrying out reactions with 750 different proteins was thus
fabricated.
EXAMPLE 2
[0103] Different oligonucleotides were synthesized by an
established method on aminosilylated silica gel particles 5 for
liquid chromatography having a diameter of 3 nm and a pore size of
10 nm.
[0104] A slurry prepared by adding an aqueous polyvinyl alcohol
solution to this oligonucleotide-immobilized silica gel was held
onto the carrier particle-immobilizing pin 7 shown in FIG. 2, and
then immobilized at a pitch 0.5 mm on the surface of a
ribbon-shaped silica gel coated polyester film 8 having a size of
approximately 0.5 cm.times.20 cm, to obtain a reactive probe chip
according to the invention.
EXAMPLE 3
[0105] Porous glass powder with a pore size of 50 nm and a diameter
of 5 .mu.m with a .gamma.-aminopropylsilylated surface was used to
synthesize different oligonucleotides by an established method.
[0106] A slurry prepared by adding an acryl polymer to the
oligonucleotide-loaded porous glass powder was held on the carrier
particle-immobilizing pin 7 shown in FIG. 3, and then arrayed and
immobilized at a pitch of 0.5 mm on the surface of an oxide
film-coated silicon chip 11 having a size of approximately 1
cm.times.1 cm, to obtain a reactive probe chip according to the
invention.
EXAMPLE 4
[0107] Porous glass powder with a pore size of 100 nm and a
diameter of 5 .mu.m with a .gamma.-aminopropylsilylated surface was
used to synthesize different oligonucleotides by an established
method.
[0108] An acryl polymer was added to each of the porous glass
powders loaded with different oligonucleotides, to prepare pastes
containing porous glass powders loaded with the different
oligonucleotides. The pastes 13 were immobilized at a pitch of 0.5
mm on the surface of a slide glass 14 (approximately 1 cm.times.1
cm), which had been subjected to a surface blasting treatment to
make the surface delustered. The immobilization was carried out by
using a multicolor screen printing technique, and this printing was
repeated (1-n) times to obtain a reactive probe chip 15 according
to the invention.
EXAMPLE 5
[0109] An enzyme with a certain specific reactivity was immobilized
on a 1 mm-square, 10 .mu.m-thick surface aminated "cover glass"
using glutaraldehyde. A "cover glass" loaded with different enzymes
was also prepared in the same manner, and these were consecutively
arranged on an acrylic adhesive-coated glass slide having a length
of 75 mm, a width of 25 mm and a thickness of 1.5 mm, using an
aligning apparatus which was a modified semiconductor wire bonder.
These were loaded into each of several 1 mm-square compartments. A
reactive probe chip capable of carrying out 100 different antigen
enzyme reactions was thus fabricated.
EXAMPLE 6
[0110] Different oligonucleotides were synthesized by an
established method on surface-aminated porous glass with a size of
0.5 mm square and a thickness of 10 .mu.m. The
oligonucleotide-immobilized porous glass was orderly arranged on an
acrylic adhesive-coated glass slide with dimensions of 75 mm
length, 25 mm width and 1.5 mm thickness. A 1000-type complementary
DNA detection chip was fabricated.
EXAMPLE 7
[0111] Different oligonucleotides were synthesized by an
established method on surface-aminated porous glass with a size of
0.5 mm square and a thickness of 10 .mu.m. The
oligonucleotide-immobilized porous glass was orderly arranged on
the surface of an epoxy adhesive-coated polyester film
(approximately 3 cm.times.20 cm, 0.3 mm thickness) at a 0.5 mm
pitch, to obtain a reactive probe chip according to the
invention.
EXAMPLE 8
[0112] As shown in FIG. 6, an alumina substrate 1 with dimensions
of 100 mm length.times.100 mm width.times.1 mm thickness was coated
with silica sol to form a silica sol layer 2, and then porous
alumina beads 3 with a mean particle size of 50 .mu.m were
dispersed thereover and dried for immobilization.
[0113] The porous alumina beads 3 had a void volume of 40% and a
surface area to weight ratio of 250 m.sup.2/g, and the individual
particles can carry agents with different functions.
EXAMPLE 9
[0114] As shown in FIG. 7, a plurality of pits 5 with dimensions of
3 mm length.times.3 mm width and 0.1 mm depth were formed by wet
etching on prescribed locations of the surface of a glass plate 4
with dimensions of 75 mm length, 25 mm width.times.1.5 mm
thickness. Polyacrylamide 6 was placed in the pits 5.
[0115] A 0.2 mm-thick porous glass plate 7 was cut into sizes
insertable in the pits 5, and the porous glass fragments 8 were
placed on the polyacrylamide 6 in the pits 5, and attached therein.
This caused a portion of the polyacrylamide 6 to spill over the
edges of the pits 5, or else the tops of the porous glass fragments
8 protruded above the surface of the glass plate 4. The attached
substrate is subjected to flattening treatment if necessary.
[0116] This method yielded a glass plate 4 having polyacrylamide 6
in a plurality of pits 5, with porous glass fragments 8 immobilized
by the polyacrylamide 6.
[0117] By dropping a function-providing reagent onto the porous
glass fragment 8 sections using a micropipette, it is possible to
manufacture a functional glass plate.
[0118] If the properties of the functional substances carried on
the porous glass fragments 8 are such that the functional
substances are affected by the polyacrylamide, the polyacrylamide
may be changed to another adhesive polymer.
EXAMPLE 10
[0119] As shown in FIG. 8, a plurality of pits 5 with dimensions of
3 mm length.times.3 mm width and 0.2 mm depth were formed by wet
etching on prescribed locations of the surface of a glass plate 4
with dimensions of 75 mm length, 25 mm width.times.1.5 mm
thickness.
[0120] After adding 2 ml of water, 4 ml of formamide and 0.2 ml of
12 N hydrochloric acid to a solution of 2 ml tetraethyl silicate in
2 ml of ethyl alcohol in a separate stirring tank, for hydrolysis
of the tetraethyl silicate, it was aged for 5 hours at room
temperature and then a highly viscous gel 10 of a porous glass
precursor was formed.
[0121] This was spin coated onto the surface of a glass plate 4 (to
0.3 mm thickness), aged and dried, to form a porous glass precursor
layer 11. Next, the porous glass precursor layer 11 sections were
polished to remove the sections raised from the surface of the
glass plate 4, to give a glass plate having a porous glass
precursor 12 only on the sections of the pits 5.
[0122] Finally, the porous glass precursor 12 was treated to
convert it to porous glass 13, to give a composite glass plate 14
having a plurality of porous glass regions in non-porous glass
regions.
EXAMPLE 11
[0123] As shown in FIG. 9, a plurality of pits 5 with dimensions of
3 mm length.times.3 mm width and 0.15 mm depth were formed by
cutting on prescribed locations of the surface of a quartz plate 15
with dimensions of 75 mm length, 30 mm width.times.1.5 mm
thickness.
[0124] Borosilicate glass particles with a mean particle size of 10
.mu.m and an oil (paraffin wax) were placed in an stirring tank 16
and stirred to form a coating solution, which was then coated onto
the surface of the quartz plate 15 by screen printing to form a
coating layer 17. The coating coverage was 400 g/M.sup.2.
[0125] This was dried and heat treated to remove the oil portion,
subsequently heated at 850.degree. C. for 30 minutes to fused
together the borosilicate glass particles, and then heat treated at
600.degree. C. for 15 hours for split-phase treatment. The
borosilicate glass sections 18 were then polished to remove the
sections raised above the surface of the glass plate 10, to give a
quartz glass plate 19 having borosilicate glass 18 only in the
sections of the pits 5.
[0126] This quartz glass plate 19 was acid treated for 0.5 hour
using sulfuric acid (1 N concentration) at approximately 90.degree.
C. to produce pores, thus giving a composite quartz glass plate 21
having a plurality of porous borosilicate glass regions 20.
[0127] According to the invention, it is possible to easily and
cheaply provide a reactive probe chip having reactive probes such
as DNA fragments integrated on its surface, without the need for
special equipment such as photolithography equipment. Also,
selection of the base material and modifications to the method of
loading the reactive probes can provide a chip with a higher degree
of integration than existing DNA chips.
[0128] By preparing a carrier loaded with different reactive
probes, it is possible to more conveniently supply chips loaded
with the necessary combinations of DNA probes for necessary
occasions, while also allowing construction of a reactive probe
chip with different types of reactive probes immobilized and
thereby providing a DNA chip of lower cost and higher stability
that can be helpful in the clinic for detection of individual DNA
polymorphisms.
[0129] According to another aspect of the invention there is
provided a composite substrate wherein a plurality of porous
regions are orderly arranged as compartments on at least a section
of the surface thereof, and therefore a plurality of different
functions or performances can be exhibited on the same substrate,
thus offering applicability not found in the prior art.
[0130] For fabrication of the composite substrate, porous glass
fragments are immobilized onto the substrate at a prescribed
spacing via an adhesive layer, or anchorable sites such as pits are
formed in the substrate at a prescribed spacing and the porous
glass fragments are immobilized therein, as a simple means of
orderly arranging a plurality of porous regions in
compartments.
* * * * *