U.S. patent application number 10/964602 was filed with the patent office on 2005-05-12 for method of producing probe arrays for biological materials using fine particles.
Invention is credited to Kambara, Hideki, Mitsuhashi, Masato.
Application Number | 20050100943 10/964602 |
Document ID | / |
Family ID | 34552995 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100943 |
Kind Code |
A1 |
Kambara, Hideki ; et
al. |
May 12, 2005 |
Method of producing probe arrays for biological materials using
fine particles
Abstract
The use of probe arrays in which probes of various biological
substances such as DNA are immobilized on the surface of a solid is
becoming established as an effective means for high-speed
screening. Different kinds of probes, such as DNA, are immobilized
on the surface of a multiple number of independently treatable fine
particles, such as beads, instead of the surface of a single solid,
and the resulting beads are aligned in a capillary or a cell in a
designated order. The size of the area where one probe is
immobilized is reduced. The bead probe array is characterized in
that such small beads are aligned one by one in a designated manner
using a sheet with holes, and one or a multiple number of beads are
held in the holes and then transferred to a probe array holder such
as a capillary.
Inventors: |
Kambara, Hideki; (Tokyo,
JP) ; Mitsuhashi, Masato; (Irvine, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
34552995 |
Appl. No.: |
10/964602 |
Filed: |
October 12, 2004 |
Current U.S.
Class: |
435/6.12 ;
427/2.11; 435/287.2; 435/6.19 |
Current CPC
Class: |
B01J 2219/00468
20130101; B01L 2300/0816 20130101; B01L 2200/0631 20130101; B01J
2219/00367 20130101; B01L 2200/12 20130101; C12Q 1/6837 20130101;
B01J 2219/00466 20130101; B01J 2219/00657 20130101; B01J 2219/00659
20130101; B01J 19/0046 20130101; B01J 2219/00337 20130101; B01L
3/5027 20130101; C40B 40/10 20130101; C40B 40/06 20130101; B01J
2219/00286 20130101; B01J 2219/00405 20130101; B01J 2219/00702
20130101; B01J 2219/00459 20130101; B01J 2219/00364 20130101; B01J
2219/00596 20130101; B01J 2219/005 20130101; B01L 2300/0636
20130101; C40B 60/14 20130101; B01J 2219/00722 20130101; B01J
2219/00308 20130101; G01N 33/54313 20130101; B01J 2219/00725
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 427/002.11 |
International
Class: |
C12Q 001/68; C12M
001/34; B05D 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2000 |
WO |
PCT/US00/09685 |
Claims
1. A method for producing a probe array, comprising the steps of:
selecting plural types of probes of interest; immobilizing the
plural types of probes on surfaces of different solid pieces; and
aligning the probe-immobilized solid pieces in a designated order
to obtain a probe array for analyzing a sample solution passing
therethrough, wherein said aligning is performed by the following
steps: (i) placing the probe-immobilized solid pieces in a well on
a sheet having a hole through which one solid piece can pass, said
hole leading to the interior of the array, said hole being closed;
(ii) trapping one of the solid pieces in said hole: (iii) opening
the hole; (iv) transferring the trapped solid piece to the array;
and (v) repeating steps (i) through (iv) until a designated number
of arrays are filled with the probe-immobilized solid pieces
aligned therein.
2. The method according to claim 1, wherein the probes are
polynucleotides, peptides, or proteins.
3. The method according to claim 1, wherein the solid pieces are
beads.
4. The method according to claim 3, wherein the beads are fine
particles.
5. The method according to claim 1, wherein the alignment of the
solid pieces is a one-dimensional arrangement or a two-dimensional
arrangement.
6. The method according to claim 1, wherein the alignment is
conducted in an array selected from the group consisting of a
capillary, a groove, and an optical cell.
7. The method according to claim 1, wherein said sheet is placed on
a movable base having a through-hole leading into the array holder,
said movable base being positioned where the hole of the sheet does
not communicate with the through-hole of the movable base; said
method further comprising after step (ii) removing the remaining
solid pieces from the sheet; and moving the movable base to a
position where the hole of the sheet communicates with the
through-hole of the movable base.
8. The method according to claim 1, wherein said hole is closed
with a valve and wherein opening said hole is performed by opening
said valve.
9. The method according to claim 1, wherein each well contains a
single type of probe-immobilized solid pieces, each well having a
hole through which one solid piece can pass, said hole being clos,
said method further comprising closing each hole after moving the
wells in a designated order to transfer each trapped solid piece to
an array; moving the wells to align the probe-immobilized solid
pieces in a next array; and repeating all steps through until a
designated number of arrays are filled with the probe-immobilized
solid pieces aligned therein.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to probe arrays for use in
detecting peptides, proteins and DNAs, diagnosing, and analyzing
biological materials including DNAs; and methods and apparatuses to
produce the same.
[0003] 2. Description of the Related Art
[0004] For DNA analyses or DNA tests or diagnoses, amplification of
a small amount of DNA, isolation and identification of the
amplified DNA fragments, and other procedures are necessary. For
DNA amplification, PCR (polymerase chain reaction) is widely used,
in which an extremely small number of DNAs can be multiplied by
several orders of magnitude so as to be detectable. On the other
hand, for the isolation and detection of different DNAs, among
other methods, a DNA sequencer and fragment analyzer, in which gel
electrophoresis and fluorescence detection are combined, are used.
However, electrophoresis becomes very labor-intensive as the number
of samples or test items increases. Thus, a simple method using DNA
probes is drawing attention, in particular, a DNA chip, in which
many kinds of probes are immobilized on the surface of a solid to
make a probe array which undergoes hybridization with the sample,
then only specific DNAs are trapped on the surface of the solid and
detected (Nature Medicine 2, 753, 1996).
[0005] The probe detection method is used also for the analysis of
proteins or peptides or various biological materials which interact
with them, and a peptide chip corresponding to the DNA chip is now
being used. This kind of isolation and detection method, in which a
peptide or DNA is immobilized on the surface of a solid and
hybridization proceeds between the peptide or DNA and a sample, has
long been known as a blotting method in which the presence of the
target DNA or the like is detected by a probe immobilized on a
membrane using radioactive labeling. However, the DNA chip, on
which a large number of probes can be immobilized on a small area
(1 cm.sup.2) of the surface of a solid such as glass or silicone,
has the advantage in that only a small amount of sample is
required, and a vast variety of probes can be used simultaneously.
Methods for the production of DNA chips are divided broadly into
two groups. In the first group, a DNA probe is synthesized one base
at a time by a photochemical reaction on small segments (0.05
mm.sup.2 to 0.2 mm.sup.2) of a solid using the same photomasking
technique as used for semiconductors or the like (Science 251, 767,
1991). In the second group, a synthesized DNA, PCR-amplified DNA,
or DNA obtained by cloning is immobilized on a small segment of the
surface of a solid for each segment of individual probes (Nature
Biotech 16, 27, 1998). The latter has the advantage that a peptide
chip or DNA chip with the required probes can be made relatively
easily, and is the method of choice of many startup companies.
SUMMARY OF THE INVENTION
[0006] A probe chip for biological materials, including DNA, is a
highly anticipated to be used as a testing tool. However, for
practical purposes, the following conditions have to be satisfied:
(A) a small amount of a large variety of chips can be made at low
cost, (B) a probe can be immobilized homogeneously, (C) data is
highly reproducible and the chip is reusable, and (D) the chip can
be heated to remove nonspecifically absorbed substances. However,
problems remain: For example, (a) the probes are not consistent
from one segment to another, (b) production is very
labor-intensive, (c) very fine segmentation for immobilization is
not possible, and (d) probes are not uniform; because (i) they are
immobilized as liquid drops on the surface of a solid, and (ii)
probes are positioned and immobilized simultaneously. Furthermore,
(d) bind weakly with the surface of the solid and may dislodged
upon heating, because (iii) many probe chips are immobilized by
adsorption or the like.
[0007] In order to solve the aforementioned problems,
immobilization of probes on the solid surface and alignment of the
probes may be separated into two or more different steps to enable
uniform DNA probes to be produced on the solid surface. The probes
can be immobilized via covalent bonds, which are heat stable,
therefore, nonspecifically absorbed substances can be appropriately
removed by heating. Fine particles, used as the solid on which
probes are immobilized, are aligned to produce a probe array having
segments of a suitable size. Any desired probe array can be readily
produced by exchanging the aligned fine particles with the probes.
Tweezers can be used to align fine particles having a diameter of
about 0.3 mm but this method would be difficult for particles
having a diameter of less than 0.1 mm. Therefore, in an embodiment,
the present invention provides a method and an apparatus to produce
a probe array, in which fine particles each held in a fine hole on
a sheet are transferred and aligned in a capillary, a groove on a
plate or the like. In an alternative method, fine particles are
controlled to flow as individual particles into a liquid for
transfer into a capillary to produce a probe array. Furthermore, in
order to improve reproducibility in measurement, a multiple number
of fine particles with a multiple number of probes are aligned for
each probe to check any variation in test results to obtain highly
reliable data.
[0008] For purposes of summarizing the invention and the advantages
achieved over the prior art, certain objects and advantages of the
invention have been described above. Of course, it is to be
understood that not necessarily all such objects or advantages may
be achieved in accordance with any particular embodiment of the
invention. Thus, for example, those skilled in the art will
recognize that the invention may be embodied or carried out in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objects or advantages as may be taught or suggested herein.
[0009] Further aspects, features and advantages of this invention
will become apparent from the detailed description of the preferred
embodiments which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] These and other features of this invention will now be
described with reference to the drawings of preferred embodiments
which are intended to illustrate and not to limit the
invention.
[0011] FIG. 1 is a conceptual view of a probe array chip comprising
beads with probes aligned in a capillary.
[0012] FIG. 2 is a conceptual view of a detection system to measure
a bead array with probes retained in a capillary or the like.
[0013] FIGS. 3a-3g are fragmentary sectional views of an apparatus
for bead alignment. FIG. 3a is a conceptual view of bead feeding in
an off-line state. FIG. 3b is a conceptual view in which a bead is
trapped in a hole. FIG. 3c is a conceptual view in which a bead is
moving into a capillary or the like. FIGS. 3d-3g show the
subsequent steps.
[0014] FIGS. 4a and 4b are conceptual views of an apparatus for the
groove-type bead alignment. FIG. 4a is a perspective illustration.
FIG. 4b is a sectional view.
[0015] FIGS. 5a, 5b, and 5c are conceptual views of a method for
producing a bead array using grooves and a movable valve. FIG. 5c
is a cross-sectional partial view.
[0016] FIG. 6 is a conceptual view of a disk-type system for probe
bead transfer.
[0017] FIG. 7 is a conceptual view of a liquid flow-type bead array
production method.
[0018] FIG. 8 is a conceptual view of a bead array in which a large
number of beads are separated with marker beads.
[0019] FIGS. 9a and 9b are conceptual views of a method of aligning
probe beads using a sheet with holes.
[0020] FIGS. 10a, 10b, and 10c are conceptual views of a microtiter
plate-type bead array holder. FIG. 10a is a general view. FIG. 10b
is a sectional view. FIG. 10c is a conceptual view for
measurement.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0021] The present invention includes a plurality of aspects and
embodiments. In one aspect, a method for producing a probe array
comprises the steps of: (a) selecting plural types of probes of
interest; (b) immobilizing the plural types of probes on surfaces
of different solid pieces, respectively; and (c) aligning the
probe-immobilized solid pieces in a designated order to obtain a
probe array for analyzing a sample solution passing therethrough.
In the above, the probes may be polynucleotides, peptides, or
proteins. In an embodiment, the solid pieces are beads which may be
fine particles. Further, the alignment of the solid pieces may be a
one-dimensional arrangement or a two-dimensional arrangement. In
another embodiment, the method further comprises placing solid
pieces as markers at specified intervals in the alignment. The
markers may have a different size from that of the solid pieces
with probes. In an embodiment, each solid piece has one type of
probe immobilized thereon, and a designated number of solid pieces
for each type of probe are prepared. Additionally, the alignment of
solid pieces may be conducted in an array selected from the group
consisting of a capillary, a groove, and an optical cell.
[0022] In an embodiment of the method, the alignment of solid
pieces may be conducted by (i) placing the probe-immobilized solid
pieces on a sheet having a hole through which one solid piece can
pass, said sheet being placed on a movable base having a
through-hole leading to the interior of the array, said movable
base being positioned where the hole of the sheet does not
communicate with the through-hole of the movable base; (ii)
trapping one of the solid pieces in the hole of the sheet; (iii)
removing the remaining solid pieces from the sheet; (iii) moving
the movable base to a position where the hole of the sheet
communicates with the through-hole of the movable base; (iv)
transferring the trapped solid piece to the array via the
through-hole; and (v) repeating steps (i) through (iv) until the
probe-immobilized solid pieces are aligned in the designated order
in the array.
[0023] In another embodiment of the method, the alignment may be
conducted by (i) placing the probe-immobilized solid pieces on a
sheet having a hole through which one solid piece can pass, said
hole leading to the interior of the array, said hole being closed
with a valve; (ii) trapping one of the solid pieces in the hole of
the sheet; (iii) opening the valve to transfer the trapped solid
piece to the array, and (iv) repeating steps (i) through (iii)
until the probe-immobilized solid pieces are aligned in the
designated order in the capillary, groove, or optical cell.
[0024] In yet another embodiment of the method, the alignment may
be conducted by (i) placing the probe-immobilized solid pieces in
wells, each well containing a single type of probe-immobilized
solid pieces, each well having a hole through which one solid piece
can pass, said hole being closed; (ii) trapping one of the solid
pieces in each hole of each well; (iii) opening and closing each
hole after moving the wells in a designated order to transfer each
trapped solid piece to an array; (iv) moving the wells to align the
probe-imrnmobilized solid pieces in a next array; and (v) repeating
steps (i) through (iv) until a designated number of arrays are
filled with the probe-immobilized solid pieces aligned therein.
[0025] In still another embodiment of the method, the alignment may
be conducted by (i) placing the probe-immobilized solid pieces in a
narrow tube; (ii) moving the solid pieces one by one with a
solution flowing along the narrow tube, to transfer the solid piece
to the array, and (iii) repeating steps (i) and (ii) until the
probe-immobilized solid pieces are aligned in the designated order
in the array.
[0026] Additionally, in an embodiment, the alignment may be
conducted by (i) placing the probe-immobilized solid pieces in
sections, each section containing a single type of
probe-immobilized solid pieces, each section having a hole through
which one solid piece can pass, said hole being closed; (ii)
trapping one of the solid pieces in each hole of each section;
(iii) opening and closing each hole after moving the sections in a
designated order to transfer each trapped solid piece to a groove;
(iv) repeating steps (i) through (iii) until the probe-immobilized
solid pieces are aligned in the groove in order; and (v)
transferring the aligned probe-immobilized solid pieces to an array
wherein the solid pieces are placed close together.
[0027] In the above, each embodiment can exhibit at least one of
the aforesaid advantageous effects.
[0028] The present invention can be applied to other aspects,
including a probe array for analyzing a sample solution passing
therethrough, and various apparatuses for manufacturing a probe
array.
[0029] The present invention will be explained by the following
examples. A probe array of the present invention can be commonly
explained either with DNAs, proteins, peptides or other biological
materials. Accordingly, DNAs are used for explanation in the
following examples.
[0030] In a DNA probe array according to the present invention,
solid probes are held either one-dimensionally in a capillary or
two-dimensionally in a small area of an optical cell. The capillary
is mainly used in the Examples for convenience of explanation.
Round beads are used as the fine particles in the Examples but any
particles having cubic or other shapes can be used. Beads having a
diameter of 1-300 microns can be used; however, beads having a
diameter of 20 microns are mainly used in the Examples. Further,
glass or plastic beads are normally used; however, metal materials
such as gold can also be used. Plastic beads are used here.
EXAMPLE 1
[0031] FIG. 1 shows an example of a probe array according to the
present invention, wherein numeral 101 is an inlet for solution and
sample, 102 is an outlet, 103 is a capillary for holding probe
array, 104 are marker beads, 105 is a bead with probe, and 106 are
dummy beads. The diameter of the beads with immobilized probes is
20 microns and the inner diameter of the capillary 103 is 25
microns. In this Example, about 20 dummy beads 106 are aligned on
both ends and 999 beads 105 are aligned between them. Every
10.sup.th bead is a black bead 104 and every 100.sup.th bead is a
red bead for a total of 99 marker beads and 900 probe beads, that
is, 900 different kinds of probes can simultaneously be used for
tests. These beads could be aligned in a 2 mm length if densely
packed; however, in this example, for hybridization and other
considerations, the beads were more loosely packed and held in a 5
mm length. The retention length can be longer or shorter than the
above described range (e.g., in the range of 2-10 mm per 1000
beads). However, an excessively long length increases the amount of
sample needed, while an excessively short length causes a problem
in handling. Moreover, a sample may be not adequately hybridized.
The volume of the reaction area is about 2.3 n liters. Stoppers are
placed in both ends to prevent the beads from flowing-out. The
sample and washing fluid are introduced and discharged through
these ends via the inlet 101 and the outlet 102. The probe array is
advantageously compact and easy to handle since as many as 10,000
probes can be held in an area of 20-30 mm in length.
[0032] The irradiating laser beam 206 and the probe holding
capillary 202 are relatively scanned and the resulting fluorescence
is measured using a fluorescence detection device, for example, as
shown in FIG. 2. In FIG. 2, numeral 201 is a bead with probe, 202
is a capillary for holding probe array, 203 is a plate to move
probe array, 204 is a point of irradiation and emission, 205 is a
lens, 206 is a irradiation laser beam, 207 is a optical filter, 208
is a lens, 209 is a laser source, 210 is a detector, 211 is a
controller for data processing and detector, and 212 is a
indicator. Different probes are readily identifiable by the aid of
marker beads placed every 10 beads 201. Marker beads can be colored
differently to identify different probes, or alternatively, each
group of 10 beads with probes can be colored differently. Of
course, in this case, colors would have to be chosen so as not to
have a wavelength which would interfere with the fluorescence
detection.
EXAMPLE 2
[0033] This Example relates to a method and an apparatus in which
beads are aligned in a capillary one at a time in predetermined
order. FIGS. 3a-3g show an example of a device to make the bead
array. In these figures, numeral 301 is an outlet for solution and
beads, 302 is an inlet for solution, 303 is a cover plate, 304 is a
bead with probe, 305 is a hole for bead trapping, 306 is a
capillary for bead alignment, 307 is a capillary holding base, 308
is a trapped bead, 309 is a nozzle for bead supply, and 310 is a
stopper. For convenience of explanation, beads are aligned in one
capillary in this Example; however, for practical use, a multiple
number of holes on a sheet and a multiple number of capillaries are
used. Step 1 (FIG. 3a): Beads with the first probe (probe bead #1,
304) are introduced with a solvent into the cell 303 having the
sheet 311 with a hole at the bottom. The beads are precipitated and
the solvent is moved back and forth and right and left to drop one
of the beads 305 into the hole. Step 2 (FIG. 3b): The remaining
beads are removed with the solvent 302 via the outlet 301 and
washed. Only the bead which dropped into the hole remains in the
cell. In this case, the solvent may be blown out of the port at a
right angle to the sheet to remove these beads near the port and
leave the one bead in the sheet hole to be introduced into the
capillary in step 3. The bottom of the hole is closed off by the
block 307. The capillary for the alignment of the beads is fixed to
this block, but in steps 1 and 2, the axis 306 of the capillary and
the hole are not aligned such that the bead 305 is retained in the
hole. Step 3 (FIG. 3c): The block 307 and the sheet 311 are moved
relative to each other to align the axis of the capillary and the
hole. Probe bead #1 (305) is introduced into the capillary by
suction from the other end or with pressure applied from the
solution injection side. In this case, the relative movement of the
block and the sheet is about the same order as the diameter of the
hole, for which a piezoelectric element is successfully used. Step
4 (FIG. 3d): The block 307 and the sheet 311 are relatively moved
so that the axis 306 of the capillary and the hole 308 are again
out of alignment. Step 5 (FIG. 3e): Beads with the second probe
(probe bead #2, 320) are introduced into the cell 303 and one of
them 321 is dropped into the hole. Step 6 (FIG. 3f): Excess beads
other than the bead in the hole are removed from the cell in the
same manner as in step 2. Step 7 (FIG. 3g): The block and the sheet
are moved relatively to align the axis of the capillary and the
hole such that the probe bead #2 (321) can be introduced into the
capillary. As a result, the bead with probe 1 (probe bead 1) and
the bead with probe 2 (probe bead 2) are aligned in the capillary.
By repeating these steps, a bead array with probes having a desired
order can be produced.
[0034] The capillary used here can be taken out and used as a probe
array holder during measurement, or a probe array holder can be
made separately and attached to the bottom part of the capillary to
which the bead array is transferred. In this Example, the probe
array holder shown in FIGS. 4a and 4b is used. In these figures,
numeral 401 is a base with a bead array holding groove, 402 is a
solution outlet capillary, 403 is an inlet for beads and various
solutions, 404 is a groove for bead alignment, 405 is a bead with
probe, 406 is a stopper, and 407 is an upper window. A sample
solution is injected from the left side (403) of the Figure. After
sufficient hybridization, a washing liquid is injected from the
right side (402) to remove the unreacted sample portion. After
mounting the probe array holder onto a measuring unit, each bead is
irradiated with a laser beam and emitted fluorescence is detected.
Of course, instead of emitted fluorescence from laser beam
irradiation, emitted light produced by a chemical emitting reagent
can also be detected. Any detection method which can detect the
presence and absence of hybridization can be used.
[0035] In this Example, the invention is explained with only one
capillary fixed to the block; however, it is possible to produce a
large number of probe arrays simultaneously using a multiple number
of capillaries. In that case, it is naturally understood that the
number of holes on the sheet has to be increased as the number of
capillaries increases.
EXAMPLE 3
[0036] This example is for an apparatus in which a bead delivery
device 504 having holes (or wells) to keep various kinds of beads
separately to transfer them to a bead arraying plate 512 having
grooves 507 on it or a capillary for aligning the beads according
to the predetermined order as a probe bead array. At first
solutions containing different kinds of probe beads placed in wells
of a titer plate are transferred one after another in a
predetermined order into designated wells (holes) of a bead
delivery device such that the beads are aligned in a groove
produced in a plate or a capillary (FIGS. 5a, 5b, and 5c). In these
figures, numeral 501 is a pipetter/injector, 502 is a titer plate
which has wells 503 containing probe beads, 504 is a bead delivery
device with holes, 505 is a hole which holds probe beads being
delivered to a groove, 506 are arrayed probe beads, 507 is a groove
in which various kinds of probe beads are aligned, 508 is a probe
bead, 509 is a probe bead trapped in a hole, 510 is a piezoelectric
element, 511 is a movable valve, and 512 is a holding base. The
beads are suctioned from the wells in the titer plate 502 with the
pipetter 501 and moved into a transfer well 505. The hole 520 for
trapping a bead is open at the bottom of the well. One of the beads
509 (a multiple number of the beads if a multiple number of the
holes are provided) injected into the well 505 drops into the hole
520, and the presence of the dropped bead is optically confirmed.
Then, excess beads are recovered or removed from the well by
flushing beads out with washing liquid. The valve 511 which can be
driven by a piezoelectric element 510 or the like is placed between
the bead trapping hole 520 and the groove 507 or the capillary. A
bead can be transferred to the groove or capillary side by moving
the valve. Actual bead movement is controlled by a liquid flow. Of
course, the bead can also be transferred by moving the plate 504 to
align the hole and the groove or the center of the capillary. Once
the bead is fully transferred, the valve is moved back or the
relative position of the hole and the capillary is shifted so that
the bead is trapped in the hole. Beads with the next probe are
introduced into the trapping site using a pipetter. The steps above
are repeated to produce a bead array. The resulting bead array 506
is used as it is, or transferred to another container while
maintaining the alignment and used as a probe array.
[0037] The steps above can be carried out in a system having a
multiple number of holes to save time in array production, or to
simultaneously produce a multiple number of the same arrays.
EXAMPLE 4
[0038] In Example 2, one kind of probe bead at a time is aligned
using a bead delivery device with one hole. In this example, a
multiple number of wells in a bead delivery device are used to
segmentally hold multiple kinds of probes bead in order to improve
productivity. As shown in FIG. 6, a multiple number of rectangular
wells 603 are placed on a rotary disk 601. In FIG. 6, numeral 601
is a disk-type bead holding plate for delivering beads, 602 is a
rotary axis, 603 is a groove for bead holding, and 604 is a hole
for bead holding. The bottom of each well is fitted with a sheet
with holes as described in Example 1 at the bottom. The lower part
of the rotary disk having the sheets has contact with a block,
which holds capillaries, to prevent dropping of the beads trapped
in the holes. When the rotary disk is moved and the holes and the
axis of the capillaries are aligned, the probe beads are
transferred into the capillaries in the same manner as described in
Example above. The number of the holes corresponds to the number of
the capillaries. The holes and the capillaries are correspondingly
positioned; however, in order to prevent shearing upon rotation, a
controlling mechanism is provided, in which the block with
capillaries is moved in the axial 602 direction of the disk using a
tracking technique similar to that used for CD-ROMs. In this
example, a rotating board having a diameter of 16 cm is used. Wells
603 (1 mm wide and 30 mm long) are located at a position 5 cm from
the axis of the disk. The pitch of the wells is 2 mm and about 150
wells can be radially placed on the disk. The sheet with holes is
spread under the wells and the pitch of the holes is 2 mm. In this
example, a total of 10 holes are aligned so that probe bead arrays
can be made in 10 capillaries. Of course, the number of capillaries
and the number of probe arrays producible at one time can be
changed as required.
[0039] The rotary plate rotates in two rotation modes; a high speed
rotation mode and a low speed but highly accurate mode. Beads are
introduced into the well with a solution. The beads are dropped
into the holes by moving the disk and flowing the solution out of
the holes. Next, excess beads are moved to bead holders located on
the end of the wells by centrifugal force and by water flow by
rotating the disk in the high speed rotating mode. The disk is
stopped, then, disk rotation is set to the highly accurate mode so
that the capillaries and the probe beads #1 align. A shutter at the
bottom of the disk is opened and the block which is holding the
capillaries is brought into contact with the rotary plate such that
the wells carrying probe bead #2 are moved to the position of the
capillaries. The beads are sequentially transferred into
capillaries to produce probe bead arrays in a designated order. A
large number of probe beads can be aligned and held in capillaries
by exchanging the disk or the probe beads to be placed in the wells
and repeating the above described steps. The position of a specific
probe in a resulting probe bead array can be conveniently confirmed
by changing the color of beads in the arrays every 10 beads.
EXAMPLE 5
[0040] This example relates to a method and an apparatus for the
alignment of probe beads into a capillary one by one in a
designated order using a liquid flow. FIG. 7 shows a conceptual
view of this example. In this figure, numeral 701 is a bead
solution reservoir, 702 is a bead with probe, 703 is a transfer
tube, 704 is a sheath flow cell, 705 is a transfer liquid, 706 is a
capillary tube for transfer, 707 is a capillary for bead array
alignment; 708 is a supporting base, and 709 is a solution outlet
tube. Beads 702 with probes are pumped into the transfer capillary
tube 707. The end of the capillary tube is inserted into a liquid
flow formed with the transfer liquid 705 in the sheath flow cell
704, and the beads are released into the liquid flow one by one,
and virtually constant intervals. However, to stabilize the
release, ultrasonic waves are applied to that portion of the
capillary holding the beads to form knots along the axis of the
capillary. The beads are released one by one into the liquid flow
at designated intervals by controlling conditions such as the
intensity of the ultrasonic waves.
EXAMPLE 6
[0041] In the examples above, one bead corresponds to one kind of
probe. However, in order to check uniformity of hybridization
reactions or to improve detection sensitivity, it is appropriate to
use a multiple number of beads for one kind of probe. It is not
necessary that the same number of beads be used for all probes. If
the number varies held in a capillary for making a probe array,
however, colored beads or beads of a different size have to be
inserted between bead groups with different probes as markers. This
example is shown in FIG. 8. In this figure, numeral 801 is a large
size dummy beads, 802 is a probe bead, 803 is a large size marker
bead, 804 is a capillary for probe holding, and 805 is a sample
flow path. The apparatus for the production is virtually the same
as described above, except that the size of the holes is several
times larger than the size of the beads 802 so that a multiple
number of beads 802 are trapped in the holes. Subsequent procedures
are the same as described above.
[0042] Further, the bead array of this example can be easily
produced if the liquid flow system described in Example 5 is used.
A small number of beads are suctioned from a bead reservoir with a
pipet and injected into the liquid flow. Although the number cannot
be confirmed, the injected beads can be sequentially placed into
the capillary 804. Prior to the injection of another kind of beads,
a colored bead or a bead of a different size (801) is injected as a
marker so that the position and the kind of probe of individual
beads can be identified.
EXAMPLE 7
[0043] The previous example is a method for the production of a
probe array in which probe beads are aligned in a capillary. This
example as shown in FIGS. 9a and 9b discloses a method and an
apparatus in which beads are first aligned in a groove produced on
a plane surface, then congregated into a probe array or transferred
into a capillary to produce a probe array. In FIGS. 9a and 9b,
numeral 901 is a plate having wells, 902 is a well for a bead
reservoir, 903 is a bead holding hole, 904 is a sheet with holes
and is usually attached to the plate 901, 905 is a base array
production holder with grooves for aligning beads, 906 is a fine
groove for probe bead alignment, 907 is a bead with probe, and 908
is a capillary for bead array. First, a bead array production
holder 905 having a multiple number of grooves 906 on a plane
surface is prepared. Beads 907 with probes are aligned in each
groove and transferred into a capillary 908 or the like while
maintaining their alignment, then the beads aligned in the multiple
number of grooves are introduced into different capillaries and
used as a probe array. A plate attached with a sheet having holes
(901, 904) is placed on top of the bead array production holder
wherein beads are trapped in the holes and transferred into the
above described grooves. As shown in FIG. 9, this plate with a
sheet has wells (bead reservoirs 902) orthogonal to the grooves on
the beads array production holder, and the holes 903 holding beads
are through holes and opened for the fine groove. The apparatus
does have a multiple number of grooves, but beads with different
probes are injected into different wells in the plate and held in
different holes. The plate attached with a sheet and the plate
having grooves are used in close contact but can slide each other.
At the start, the holes 903 of the sheet and the grooves 906 for
the bead array production holder are not aligned. Beads with probes
are supplied into different wells 902 of the plate above the sheet
with holes for each kind of probe. One bead drops into one hole and
retained there because the bottom of the hole is closed at this
state. When the holes of the sheet and the grooves of the bead
array production holder are aligned, the beads drop one by one from
individual holes into the grooves 906. Since different probe beads
drop into one groove from different positions, a variety of probe
beads are retained in a groove. The beads are placed virtually at
the same intervals as those in the bead reservoir 902 on the sheet
with holes. In this example, the interval is 2 mm. A total of 50
beads are dropped into each groove of the bead array production
holder in this example. Also, 10 bead arrays can be simultaneously
produced in this example, but the number can be increased as
desired. After dropping the beads, the positions of the sheet with
holes 903 and the grooves 906 of the bead array production holder
are shifted to seal up the grooves, after which the beads are
introduced into the capillary 908 with a liquid flow. The number of
different kinds of probes can be arrayed by repeating the above
described steps.
[0044] In this example, a one-dimensionally aligned probe bead
array is disclosed; however, naturally, probe arrays having many
more kinds of probes can be produced by arranging a multiple number
of these arrays or by two-dimensionally aligning these arrays.
EXAMPLE 8
[0045] In this example, a probe bead array holder comprises cells
which consist of a plate with one-dimensionally or
two-dimensionally distributed holes and a cover glass. In FIG. 10a,
10b, and 10c, numeral 1001 is a microtiter plate-type bead array
holder, 1002 is a spacer, 1003 is hole which makes a bead array
cell with a cover glass, 1004 is a bead with probe, 1005 is a cover
glass, 1006 is a solution outlet, 1007 is a solution inlet, 1008 is
a laser beam, 1009 is a lens, and 1010 is a detector. This
resembles a micro titer plate. A small number of beads are
suctioned from a titer plate in which probe beads are held, and
dispensed into holes (cells) 1003 of the plate 1001. The beads 1004
are dispensed into the holes at designated positions according to
the kind of probe to produce a microtiter plate-type bead array
with probe beads. After the beads are dispensed, the cover glass
1005 which is optically transparent and does not interfere with the
measurement of fluorescence or chemical emission is placed on top
to produce a cell array. The space between the cover glass and
walls, which segment the cells of the microtiter plate-type cell
array, is smaller than the size of the beads so that the beads
cannot move out. The reaction solution or the like can flow through
the cells freely. For use, the cells are turned upside down to make
the glass side down. In this case, the beads on the glass surface
are sufficiently in contact with the reaction solution independent
of the depth of the cells and the probes undergo hybridization with
the target.
[0046] [Effectiveness of the Invention]
[0047] As described above, according to the present invention, a
large number of probe arrays for peptides or DNAs can be produced
by a simple procedure. The process to immobilize probes on the
surface of a solid and the process to align probes are separated,
so that both processes can be optimized. As a result, immobilized
probes which are homogeneous and not easily removable from the
surface of the solid can be produced, then an array having the
required kinds of probes can be readily produced by aligning the
beads in a designated order. Also, a fine probe array, which is
difficult to make by a conventional method, can be produced by
reducing the size of the beads. A probe array with new components
can be produced simply by preparing the required DNA probes,
immobilizing them on the surface of beads and setting the probe
beads onto a production apparatus, and thus arrays as requested by
users can be provided any time. By aligning a multiple number of
beads carrying the same probes, statistical averages can be
obtained to analyze reproducibility and quantitativeness, and
reliable measurements can be carried out. Furthermore, the reaction
is quick and highly sensitive because the surface area for the
reaction is larger than that in conventional DNA chips or the like
being retained on a plane. The size of the beads can vary between 1
micron to 300 microns so that high density probe arrays can be
readily produced if necessary. For example, by using 6-micron
beads, 1,500 probes can be aligned in a 10-mm length in a
capillary, or more than 1,000,000 probes can be retained in an area
of 1 cm.sup.2 if a two-dimensional probe array holder is used.
[0048] A multiple number of arrays having the same probe alignment
can be produced by an extremely simple procedure and thus the
arrays are also suitable for mass production.
[0049] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
* * * * *