U.S. patent application number 10/105303 was filed with the patent office on 2002-10-17 for probe carrier and method of manufacturing the same.
Invention is credited to Mihashi, Naoto, Yamamoto, Nobuko.
Application Number | 20020150942 10/105303 |
Document ID | / |
Family ID | 18947623 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150942 |
Kind Code |
A1 |
Yamamoto, Nobuko ; et
al. |
October 17, 2002 |
Probe carrier and method of manufacturing the same
Abstract
A flat plate-shaped probe carrier for carrying probes such as
single stranded DNAs, proteins, etc. which are reactive
specifically with target substances comprises a plurality of ring
bodies arranged substantially in parallel and the external space
among the ring bodies is filled with a filler such that the
openings of the ring bodies are oriented to the surface of the
probe carrier. Each ring body has a region for fixing a probe
adapted to be bonded specifically to a target substance on its
inner wall. The probe carrier is produced by bundling a plurality
of hollow tubular members in parallel, then filling the external
space among the bundled hollow members with a filler, and cutting
the bundle along a plane intersecting the axial direction of the
tubular members. Probes are fixed to the fixing region before or
after filling the external space.
Inventors: |
Yamamoto, Nobuko; (Kanagawa,
JP) ; Mihashi, Naoto; (Tokyo, JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
18947623 |
Appl. No.: |
10/105303 |
Filed: |
March 26, 2002 |
Current U.S.
Class: |
435/6.19 ;
427/2.11; 435/287.2 |
Current CPC
Class: |
Y10T 156/1052 20150115;
Y10T 436/143333 20150115; B01J 2219/00673 20130101; C40B 60/14
20130101; B01J 2219/00421 20130101; B01J 2219/00722 20130101; B01J
2219/00317 20130101; B01J 2219/00621 20130101; B01J 2219/00605
20130101; B82Y 30/00 20130101; C40B 40/06 20130101; B01J 2219/00677
20130101; B01J 19/0046 20130101; B01L 3/5085 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 427/2.11 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2001 |
JP |
093268/2001(PAT) |
Claims
What is claimed is:
1. A flat plate-shaped probe carrier comprising a plurality of ring
bodies having in the inside a region for fixing a probe adapted to
be bonded specifically to a target substance and a filler filled
among said ring bodies without any substantial space left
therein.
2. A probe carrier according to claim 1, wherein said target
substance is a nucleic acid and said probe has a sequence
complementary relative to part or all of the base sequence of said
nucleic acid and is a single stranded nucleic acid adapted to be
specifically hybridized with said nucleic acid.
3. A probe carrier according to claim 1, wherein said ring bodies
have respective probes of different types in the inside.
4. A method of manufacturing a probe carrier comprising a plurality
of ring bodies having in the inside a region for fixing a probe
adapted to be bonded specifically to a target substance, said
method comprising; a step of bundling a plurality of hollow tubular
members to make them arranged substantially in parallel with each
other; a step of filling the external space among the bundled
hollow tubular members with a filler and solidifying the filler; a
step of cutting the bundle of the tubular members filled with the
filler along a plane intersecting the axial direction of the
tubular members to produce a flat plate-shaped carrier comprising a
plurality of ring bodies having respective internal openings; and a
step of fixing probes to the respective regions for fixing a probe
before or after filling the space with the filler.
5. A method according to claim 4, wherein said target substance is
a nucleic acid and said probe has a sequence complementary relative
to part or all of the base sequence of the nucleic acid and is a
single stranded nucleic acid adapted to be specifically hybridized
with the nucleic acid.
6. A method according to claim 4, wherein said filler is in a gel
state before said step of filling the external space and the
external space of the tubular members is filled with the filler by
applying centrifugal force to the filler, wherein the filler flows
in a sol state which centrifugal force is being applied.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a probe carrier carrying a probe
rigidly secured thereto for detecting a target substance. The
present invention also relates to a method of manufacturing such a
probe carrier.
[0003] 2. Related Background Art
[0004] When analyzing the base sequence of a gene DNA or conducting
a gene diagnosis by analyzing a number of items simultaneously,
probes of different types are needed to single out a DNA having a
target base sequence in order to raise the reliability of
operation. DNA microchips have been attracting attention as means
for providing probes of a number of different types to be used for
such sorting operations. A large number of solution species (e.g.,
96, 384 or 1,536 species) containing proteins or drugs to be sorted
normally have to be subjected to a screening operation in an
orderly manner in the field of high throughput screening of
chemicals or (combinatorial chemistry. For these purposes,
techniques of sequentially arranging a large number of different
types of drugs, automatic screening technologies and dedicated
devices for sorting the drugs arranged in this way and software for
controlling a number of screening operations and statistically
processing the obtained results have bean and being developed.
[0005] Basically, such screening operations as described above that
are conducted in parallel simultaneously consist in detecting an
action or non-action or a response or non-response of each specimen
to the known probes arranged in array, or probe array, provided as
means for sorting the substances of specimens for evaluation under
same conditions. Generally, the action or response to be used with
each probe is defined in advance and therefore substances of a same
type are normally used as probe species that are mounted on a probe
array. Then, the probe array may be that of DNA probes carrying a
group of DNAs having different respective base sequences. DNAs,
proteins and synthesized chemicals (drugs) are examples of
substances that may be used for a group of probes. While a probe
array of a group of a plurality of probe species is used in many
instances, a large number of identical DNAs having a same base
sequence, identical proteins having a same amino acid sequence or
identical chemical substances may be arranged in array. Such probes
are mainly used for screening drugs. If the target substance is a
nucleic acid, the probes may be formed by using a single stranded
nucleic acid that shows a complementary sequence relative to all or
part of the target nucleic acid and is adapted to a specific
hybridization with the base sequence of the target nucleic
acid.
[0006] In a probe array formed by a plurality of probe species, a
group of DNAs having different base sequences, a group of proteins
having different amino acid sequences, a group of different
chemical substances or the like are often arranged in array on a
substrate according to a predetermined sequence of arrangement
Particularly, DNA probe arrays are used for analyzing the base
sequence of a gene DNA or conducting a gene diagnosis by analyzing
a number of items simultaneously in order to raise the reliability
of operation as pointed out above.
[0007] U.S. Pat. No. 5,424,186 describes a technique of preparing
an array of DNA probes with DNAs having respective base sequences
that are different from each other by means of a stepwise
elongation reaction conducted on a carrier by utilizing
photodecomposable protective groups and photolithography. With the
proposed technique, it is possible to prepare a DNA probe array
carrying DNAs of more than 10,000 different kinds that are
different from each other in terms of base sequence per 1 cm.sup.2.
The process of synthesizing a DNA by means of a stepwise elongation
reaction, using this technique, comprises a photolithography step
in which dedicated photomasks are used respectively for the four
different kinds of base (A, T, C, G) in order to selectively
elongate any of the bases at a predetermined position of the array
so that consequently DNAs of different species having desired
respective base sequences are synthetically produced and arranged
on a substrate in a predetermined order.
[0008] Beside the above described technique, techniques of
manufacturing a probe array by synthesizing DNAs for probes in
advance in a refined manner, confirming, if necessary, their
respective base lengths and supplying the DNAs to a substrate by
means of an appropriate device such as a microdispenser are known.
PCT Patent Publication WO95/35505 describes a technique of
supplying DNAs onto a membrane by means of capillaries. With this
technique, it is theoretically possible to manufacture about 1,000
DNA arrays per 1 cm.sup.2. It is basically a technique of
manufacturing a probe array by supplying a probe solution to a
predetermined position of a substrate for each probe by means of a
capillary-shaped dispensing device and repeating this
operation.
[0009] There are also known techniques of supplying a solution of a
substance necessary for conducting an operation of DNA solid phase
synthesis on a substrate in each elongation step. For example,
European Patent Publication EP0703825B1 describes a technique of
synthesizing DNAs of a plurality of different species having
respective predetermined base sequences in a solid phase by
supplying nucleotide monomers and activators by means of respective
piezo jet nozzles for the purpose of solid phase synthesis of DNAs.
This supply (application) technique utilizing an ink-jet method is
reliable in terms of reproducibility of supply rate if compared
with a solution supply (application) technique utilizing
capillaries and also provides advantages for realizing high density
probe arrays because the nozzle structure of the ink-jet system can
be miniaturized.
[0010] Patent Publication EP895082 discloses a technique of causing
liquid containing a probe to adhere to a solid phase as liquid
droplets by means of a bubble jet head and forming a spot
containing a probe on the solid phase. Patent Publication
WO00/53736 discloses a method of manufacturing arrays by
immobilizing nucleic acid in a hollow thread.
[0011] Thus, it is the object of the present invention to provide a
probe carrier that is adapted to mass production and a method of
Manufacturing such a probe carrier.
SUMMARY OF THE INVENTION
[0012] In an aspect of the invention, there is provided a flat
plate-shaped probe carrier comprising a plurality of ring bodies
having in the inside a region for fixing a probe adapted to be
bonded specifically to a target substance and a filter filled among
the ring bodies without any substantial space left therein.
[0013] In another aspect of the invention, there is provided a
method of manufacturing a probe carrier comprising a plurality of
ring bodies having in the inside a region for fixing a probe
adapted to be bonded specifically to a target substance, the method
comprising:
[0014] a step of bundling a plurality of hollow tubular members to
make them arranged substantially in parallel with each other;
[0015] a step of filling the external space among the bundled
hollow tubular members with a filler and solidifying the
filler;
[0016] a step of cutting the bundle of the tubular members filled
with the filler along a plane intersecting the axial direction of
the tubular members to produce a flat plate-shaped carrier
comprising a plurality of ring bodies having respective internal
openings; and
[0017] a step of fixing probes to the respective regions for fixing
a probe before or after filling the space with the filler.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A and 1B are schematic illustrations of a probe
carrier according to the invention.
[0019] FIG. 2A is a schematic illustration of an apparatus for
forming a region for fixing a probe in a hollow member.
[0020] FIG. 2B is a schematic illustration of a region for fixing a
probe.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] Each region for fixing a probe in a probe carrier according
to the invention is formed on the inner wall of the opening section
of a ring body. FIGS. 1A and 1B schematically illustrate a probe
carrier according to the invention and having such a configuration.
FIG. 1A is a schematic plan view of the probe carrier, showing the
surface thereof having opening sections 2a of the ring bodies 2.
FIG. 1B is a schematic cross sectional view of the probe carrier
taken along line 1B-1B in FIG. 1A.
[0022] Referring to FIGS. 1A and 1B, the probe carrier 1 has a
planar surface where the openings of the opening sections 2a of the
ring bodies 2 are arranged. A probe is fixed to the surface of the
inner surface 2c of each opening section 2a. The region for fixing
a probe of each ring body 2 is formed by the inner surface 2c of
the opening section 2a.
[0023] The ring bodies 2 can be formed by using fiber, glass, resin
or some other similar material.
[0024] From the viewpoint of efficiently producing a large number
of ring bodies having probes fixed thereto, it is preferable to fix
probes to the insides of respective tubular members, or hollow
members, whose hollow areas are extending axially, and subsequently
cut the tubular members in a direction intersecting the axial
direction to obtain a large number of hollow pieces, or ring
bodies, having a predetermined height. In the instance illustrated
in FIGS. 1A and 1B, each ring body has a pair of opposite surfaces
produced by truncation and running substantially in parallel with
each other in a direction perpendicular to the axial direction of
the hollow members. At least one of the opposite surfaces can be
used for arranging a region for fixing a probe of the carrier. The
operation of fixing a probe to the hollow area of each tubular
member of the probe carrier may come before or after the operation
of filling the outside space of the tubular members with a filler
and solidifying it.
[0025] The external space of the ring bodies of the probe carrier
is filled with a filler 3 to produce a layer 4 comprising the ring
bodies as integral parts thereof. While the layer 4 is laid on a
base member 5 supporting the former in the instance of FIGS. 1A and
1B, the base member 5 may be omitted if the layer 4 itself can
provide a satisfactory mechanical strength.
[0026] For the purpose of the present invention, the expression of
"without any substantial space left therein" refers to a state
where the space is filled with the filler to such an extent that
cleansing or specimen solutions and various reagents would not
permeate after washing the ring carrier.
[0027] A probe carrier as shown in FIGS. 1A and 1B can be
manufactured by a method as described below.
[0028] Firstly, the inside of each hollow member that may be a
tubular member or a hollow thread is filled with a solution
containing a probe and the probe is fixed to the inner wall of the
hollow member.
[0029] The hollow member may be formed typically by extrusion
molding of polypropylene film that is used for purification of
water, artificial lung and artificial liver, polyethylene film or
cupro-ammonia cellulose regenerated film.
[0030] The above fixing operation is conducted for each probe
necessary for forming the probe carrier. If necessary, a reagent to
be used for the purpose of fixation and/or a cleansing solution is
supplied to the inside of the hollow member. Any known appropriate
technique can be used for the fixing operation. For example, liquid
containing the probe may be poured into the hollow area of the
hollow member to make the inner wall of the hollow member and the
probe contact and react with each other. The probe may be fixed to
the inner wall of the hollow member by means of a chemical reaction
utilizing any of various reaction groups and/or physical
adsorption, which will be described hereinafter. A hollow member
carrying the probe fixed to the inner wall thereof is obtained by
appropriately cleansing the hollow of the hollow member.
[0031] The hollow member now carrying the probe fixed to the inner
wall thereof is closed at an end. Then, a necessary number of
similar hollow members are arranged in a container to be used for a
centrifugal process in parallel with each other along the axial
direction thereof and bundled with the ends made flush with each
other. Note that the hollow members are arranged and bundled in
such a way that, when they are cut to show opposite surfaces, the
region of each hollow member where the probe is fixed is located at
a predetermined position on the corresponding surface.
[0032] FIG 2A shows how the hollow members 7 are arranged in a
container 6 that is to be used for a centrifugal process. Under
this condition, the container 6 is made to turn along a circular
track having a given radius of gyration with the central axis of
the container 6 located on a radius of gyration in order to
generate centrifugal force that is directed toward the end of the
bundle of the hollow members in the direction of the arrow shown in
FIG. 2A. A bonding material (potting material) that may typically
be polyurethane is driven to move in the direction of the arrow and
fill the external space of the hollow members 7 by the centrifugal
force. As the application of the centrifugal force is suspended,
the mobilized bonding material is solidified so that a fixed
section 8 as shown in FIG. 2B is obtained. If the bonding material
is in a gel state and subjected to centrifugal force, shearing
force is generated in the bonding material to make it have a
thixotropic property and turn into a sol state. As a result, the
filler is mobilized and driven to move into the external space of
the tubular members.
[0033] The ends of the hollow members where the fixed section 8 is
formed is preferably closed by (filled with) resin if the lateral
wall separating the hollow and the external space of each hollow
member shows gas permeability. If the lateral wall of each hollow
member does not show gas permeability, it is preferable to close
the end where the fixed section 8 is formed in order to reliably
prevent the bonding material (filler) from flowing into the hollow
of the hollow member.
[0034] The gyration of the container 6 is terminated when the
solidification of the bonding material is completed. Then, the
hollow members 7 are cut into a short piece at predetermined
positions of the fixed section 8 along a plane perpendicularly, for
example, intersecting the axial direction of the hollow members
that are arranged in parallel with and rigidly secured to each
other. The plane of arrangement of the hollow ring bodies 2 having
respective openings is made to agree with one of the opposite
surfaces of the short piece as shown in FIGS. 1A and 1B. In other
words, the ring bodies 2 illustrated in FIGS. 1A and 1B are formed
by truncating the hollow members 7 to show a predetermined height A
probe carrier having regions for fixing probes that are arranged in
a predetermined manner is obtained from the short piece.
[0035] With the above described method of manufacturing a probe
carrier according to the invention, it is possible to manufacture a
large number of probe carriers having a same and identical
configuration by cutting a number of hollow members at a fixed
section to produce short pieces. Additionally, the external space
of the ring bodies is filled with a bonding material because, if
the space is not filled and left as gaps, problems may arise as a
specimen solution containing the target substrate and various
reagents flow into the space.
[0036] According to the invention, the probes fixed to the probe
carrier can be specifically bonded to a specific target substance.
Additionally, the probes may contain oligonucleotide,
polynucleotide and/or other polymers that can recognize a specific
target. The term of "probe" as used herein refers to a group of
molecules that can operate as probe such as polynucleotide
molecules or that of molecules such as polynucleotide molecules
that are fixed to distributed respective positions of a surface to
show a same and identical arrangement and includes molecules called
ligands. The probe and the target can often be exchangeable and
also can he bonded or made to become bonded to each other as part
of ligand-antiligant (or receptor) pairs. For the purpose of the
invention, a probe and a target can contain one or more than one
natural bases and/or analogs.
[0037] Examples of probe that can be supported on a carrier include
a substance having a bonding section that is to be bonded to the
carrier in a part of the oligonucleotide thereof showing a base
arrangement that can be hybridized with a target nucleic acid, said
bonding section to be bonded to the carrier will show a structure
linked to the surface of the carrier. There are no limitations to
the position of the bonding section to be bonded to the carrier in
the oligonucleotide molecule so long as the desired reaction of
hybridization is not adversely affected.
[0038] While the probes to be carried by a probe carrier
manufactured by a method according to the invention may be
appropriately selected depending on the application thereof, they
are preferably selected from DNAs, RNAs, cDNAs (complementary
DNAs), PNAs, oligonucleotides, polynucleotides and other nucleic
acids, oligopeptides, polypeptides, proteins, enzymes, substrates
relative to enzymes, antibodies, epitopes relative to antibodies,
antigens, hormones, hormone receptors, ligands, ligand receptors,
oligosaccharides and polysaccharides for the purpose of
advantageously embodying the method of the present invention. If
necessary, two or more than two of the above listed substances may
be combined for use.
[0039] For the purpose of the invention, a probe carrier refers to
an object realized by fixing a plurality of probe species in
independent respective regions of the surface of the carrier
(including the surface of the inner walls of hollow members or
tubular carrier members) typically as dot-shaped spots, while a
probe array is one where such respective regions are arranged at a
predetermined interval.
[0040] On the other hand, each probe has a structure that makes it
possible to be bonded to the surface of the carrier and preferably
be bonded onto the carrier by way of the structure that makes it
possible to be bonded to the surface thereof. Preferably, the
structure of each probe that makes it possible to be bonded to the
surface of the carrier is formed by means of a process of
introducing an organic functional group such as an amino group, a
mercapto group, a carboxyl group, a hydroxyl group, an acid halide
(haloformyl group; --COX), a halide (--X), aziridine, a maleimide
group, a succinimide group, isothiocyanate, a sulfonylchloride
(--SO.sub.2Cl) group, an aldehyde group (formyl group, --CHO),
hydrazine or acetamide iodide. Whenever necessary, the surface of
the carrier may be treated appropriately in a manner that depends
on the structure necessary for bonding the probes to the
carrier.
* * * * *