U.S. patent application number 10/098330 was filed with the patent office on 2002-10-03 for capillary array and capillary array photodetector.
Invention is credited to Inaba, Ryoji, Kita, Toshiaki, Kojima, Masaya, Morioka, Tomonari, Ozawa, Miho, Shimizu, Yasushi, Suzuki, Akihiro.
Application Number | 20020140934 10/098330 |
Document ID | / |
Family ID | 18956294 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020140934 |
Kind Code |
A1 |
Inaba, Ryoji ; et
al. |
October 3, 2002 |
Capillary array and capillary array photodetector
Abstract
A multi-focus capillary array that can reduce crosstalk and a
capillary array photodetector are provided. On a substrate 20 on
which a plurality of capillaries are aligned, a perforation 72 is
formed in at least a part of an area in the rear of the capillaries
when the capillary array is viewed from the photodetector.
Inventors: |
Inaba, Ryoji; (Hitachinaka,
JP) ; Kita, Toshiaki; (Hitachinaka, JP) ;
Ozawa, Miho; (Abiko, JP) ; Morioka, Tomonari;
(Hitachinaka, JP) ; Suzuki, Akihiro; (Hitachinaka,
JP) ; Shimizu, Yasushi; (Hitachinaka, JP) ;
Kojima, Masaya; (Mito, JP) |
Correspondence
Address: |
DICKSTEIN SHAPIRO MORIN & OSHINSKY LLP
2101 L STREET NW
WASHINGTON
DC
20037-1526
US
|
Family ID: |
18956294 |
Appl. No.: |
10/098330 |
Filed: |
March 18, 2002 |
Current U.S.
Class: |
356/344 ;
204/452; 204/603 |
Current CPC
Class: |
G01N 27/44721 20130101;
G01N 27/44782 20130101 |
Class at
Publication: |
356/344 ;
204/603; 204/452 |
International
Class: |
G01N 027/26; G01N
027/447 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 2001 |
JP |
2001-103206 |
Claims
What is claimed is:
1. A capillary array comprising a substrate having a planar
capillary holding surface and a plurality of capillaries aligned on
the capillary holding surface of the substrate, wherein a laser
beam irradiated on a capillary at one end or capillaries at both
ends of the plurality of capillaries in a substantially parallel
direction with respect to the capillary holding surface propagates
to all the plurality of capillaries one by one to travel through
the capillaries, and emissions from each of the capillaries are
detected in a substantially perpendicular direction with respect to
the capillary holding surface, wherein the substrate is provided
with a through hole extending from the capillary holding surface to
a back side of the substrate in an area opposing to portions of the
plurality of capillaries each of which receives the laser beam
irradiation.
2. A capillary array comprising a substrate having a planar
capillary holding surface and a plurality of capillaries aligned on
the capillary holding surface of the substrate, wherein a laser
beam irradiated on a capillary at one end or capillaries at both
ends of the plurality of capillaries in a substantially parallel
direction with respect to the capillary holding surface propagates
to all the plurality of capillaries one by one to travel through
the capillaries, and emissions from each of the capillaries are
detected in a substantially perpendicular direction with respect to
the capillary holding surface, wherein the capillary holding
surface of the substrate opposing to portions of the plurality of
capillaries each of which receives the laser beam irradiation is
subjected to a light-scattering prevention treatment or a
light-reflection prevention treatment.
3. A capillary array comprising a substrate having a planar
capillary holding surface, a plurality of capillaries aligned on
the capillary holding surface of the substrate, scattered light
shielding means placed on the plurality of capillaries for
shielding areas at which capillaries contact with each other with a
partial area including are adjacent to one another with partial
areas including a central axis of each of the capillaries being not
shielded, wherein a laser beam irradiated on a capillary at one end
or capillaries at both ends of the plurality of capillaries in a
substantially parallel direction with respect to the capillary
holding surface propagates to all the plurality of capillaries one
by one to travel through the capillaries, and emissions from each
of the capillaries are detected through the space between the
shielding means in a substantially perpendicular direction with
respect to the capillary holding surface, wherein each of the
capillaries is provided with a coating, but is not provided with
the coating in an area for receiving the laser beam irradiation
thereon, and the scattered light shielding means contacts with the
area of the capillaries for which the coating is not provided.
4. A capillary array photodetector comprising a reference surface
which contacts with a capillary holding surface of a capillary
array holding substrate that holds a capillary array, fixing means
for fixing the capillary array holding substrate brought into
contact with the reference surface by pressing the capillary array
holding substrate from the rear surface thereof, a laser light
source, an irradiation optical system for setting a part of a light
path extending from the light source to be substantially parallel
to the capillary holding surface of the capillary array holding
substrate contacting with the reference surface, and a
photodetector system for detecting emissions, the capillary array
photodetector detecting emissions from each of the capillaries,
which emissions being caused by laser beam that is irradiated from
the irradiation optical system on a capillary at one end or
capillaries at both ends of the plurality of capillaries aligned on
the capillary holding surface of the capillary array holding
substrate fixed on the reference surface with being brought into
contact therewith and that propagates to all the plurality of
capillaries one by one to travel through the capillaries, wherein
the fixing means is provided with a recessed portion opening on a
side of the reference surface.
5. The capillary array photodetector according to claim 4, wherein
a surface of the recessed portion of the fixing means is subjected
to a light-scattering prevention treatment or a light-reflection
prevention treatment.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a capillary array
electrophoresis device for separating a sample such as fluorescence
labeled DNA and detecting, identifying and analyzing a base
sequence, a base length and so on of the sample according to
electrophoresis in a capillary, and more particularly, to a
capillary array to be incorporated into the capillary array
electrophoresis device and a capillary array photodetector for
detecting emission from the sample migrating through the capillary
array.
[0002] Capillary electrophoresis has been utilized for
determination of DNA base sequences and DNA base lengths. In the
capillary electrophoresis, a sample containing DNA as a measuring
object is injected into a capillary made of glass or the like and
filled with a polyacrylamide gel or the like, and then a voltage is
applied to each of the ends of the capillary. The synthetic DNA in
the sample migrates through the capillary to be separated into
fragments according to molecular weights thereof and so on, and
then a DNA band is generated in the capillary. A fluorescent dye
molecule is bonded to each of the synthetic DNA fragments, and
emission measuring means measures emission from each of the
fragments by way of laser beam irradiation, to thereby determine
base sequences, base lengths and the like of the synthetic DNA
fragments from the measured fluorescent spectra.
[0003] U.S. Pat. No. 5,582,705 discloses a multi-focus system,
which is a variation of a system of irradiating a plurality of
capillaries with light. In the system, laser beam is irradiated on
a capillary at one end or capillaries on both ends of a capillary
array comprising a plurality of capillaries aligned in parallel to
each other on a planar substrate, and the laser beam is propagated
to adjacent capillary or capillaries one by one to ultimately
travel across the capillary array so that a photodetector detects
emissions generated in the capillary array. Each of the capillaries
is applied with a polymer coating, but the polymer coating is not
applied at part of the capillaries to be irradiated with a laser
beam.
[0004] There have been proposed irradiation systems other than the
multi-focus system, such as a scanning system wherein a plurality
of capillaries to scan each of the capillaries is irradiated with a
laser beam (Nature, 359 (1992)), a multi-beam system wherein each
of a plurality of capillaries is irradiated with a laser beam
(Analytical Chemistry, 65, 956 (1993)), and a batch irradiation
system (Analytical Chemistry, 66, 1424 (1004)) wherein a plurality
of capillaries are subjected to a batch-irradiation with a laser
beam that is spread by a cylindrical lens in a direction along
which the capillaries are aligned. The multi-focus system has the
advantage of excellent detection sensitivity for DNAs as compared
with the above systems.
SUMMARY OF THE INVENTION
[0005] In the multi-focus system, it is necessary to suppress a
relative misalignment among a plurality of capillaries as small as
possible in order to propagate a laser beam through the
capillaries. Therefore, an alignment precision required for the
system is typically realized by fixing the capillaries on a planar
glass substrate by so pressing them that the adjacent capillaries
contact with one another. Since the laser beam passes through the
capillaries thus brought into contact with one another, emissions
of and from the laser beam and capillaries are reflected and
scattered complicatedly on a surface of each of the capillaries.
Further, the scattered light becomes more complicated due to the
planar substrate on which the capillaries are aligned.
[0006] There is a problem of "crosstalk" in a multi-focus system
that a part of emissions from a certain capillary overlaps with a
location of emissions of an adjacent capillary due to the scattered
light, i.e., signals of the certain capillary are detected as
signals of the adjacent capillary.
[0007] In view of the problem in the art, an object of the present
invention is to provide a multi-focus type capillary array and a
capillary array photodetector that allow reduction of the
crosstalk.
[0008] In order to achieve the object, the present invention
provides a capillary array comprising a substrate having a planar
capillary holding surface and a plurality of capillaries aligned on
the capillary holding surface of the substrate, wherein a laser
beam irradiated on a capillary at one end or capillaries at both
ends of the plurality of capillaries in a substantially parallel
direction with respect to the capillary holding surface propagates
to all the plurality of capillaries one by one to travel through
the capillaries, and emissions from each of the capillaries are
detected in a substantially perpendicular direction with respect to
the capillary holding surface, wherein the substrate is provided
with a perforation piercing from the capillary holding surface to a
back side of the substrate in an area opposing to portions of the
plurality of capillaries each of which receives the laser beam
irradiation.
[0009] It is preferable to secure a possibly largest space free
from such an object that can cause the emissions from the
capillaries to reflect to reach the photodetector on the rear side
of each of the capillaries when the capillary array is viewed from
a photodetector for detecting the emissions from the capillaries.
Since the substrate is provided with the perforation, the emissions
from the capillaries are no longer reflected from the substrate and
does not enter the photodetector, thereby reducing the
crosstalk.
[0010] In conventional irradiation systems other than the
multi-focus system, there has been proposed a variation wherein a
wide area is secured on the rear side of capillaries when a
capillary array is viewed from a photodetector. For example, "Mega
BACE", a capillary electrophoresis device manufactured by Molecular
Dynamics, Inc. is provided with such area. However, since the
device does not employ the multi-focus irradiation, the device does
not require such a high level of relative alignment precision as
that required in the multi-focus system. The present invention is
distinguished from the above-mentioned systems in the ability to
reduce the crosstalk while suppressing relative misalignment among
the capillaries to a remarkably low level.
[0011] Further, in order to achieve the above object, the present
invention provides a capillary array comprising a substrate having
a planar capillary holding surface and a plurality of capillaries
aligned on the capillary holding surface of the substrate, wherein
a laser beam irradiated on a capillary at one end or capillaries at
both ends of the plurality of capillaries in a substantially
parallel direction with respect to the capillary holding surface
propagates to all the plurality of capillaries one by one to travel
through the capillaries, and emissions from each of the capillaries
are detected in a substantially perpendicular direction with
respect to the capillary holding surface, wherein the capillary
holding surface of the substrate opposing to portions of the
plurality of capillaries each of which receives the laser beam
irradiation is subjected to a light-scattering prevention treatment
or a light-reflection prevention treatment.
[0012] Here, the light-scattering prevention treatment or the light
reflection prevention treatment may be a monolayer or a multilayer
anti-reflection coating. Conventionally, a groove is formed on a
planar substrate along a laser light path in order to avoid contact
of the laser beam with the planar substrate on which capillary
array is formed and, therefore, the glass surface is frosted as it
is a frosted glass as a result of the grooving. Elimination of such
groove in the rear area of the capillaries when the capillary array
is viewed from the photodetector contributes to prevention of the
light scattering. That is to say, the light-scattering prevention
treatment includes forming a simply flat surface in place of the
frosted glass-like surface. A groove for avoiding contact of laser
beam with the substrate may be formed only on each of ends of the
glass substrate so that the groove is not necessary in the rear
area of the capillaries when the capillary array is viewed from the
photodetector. Further, it is possible to avoid contact of the
laser beam with the planar substrate by downsizing the planar
substrate or shifting an irradiation angle of the laser beam
parallelly with respect to the planar substrate without forming the
groove on the planar substrate.
[0013] Further, in order to achieve the above object, the present
invention provides a capillary array comprising a substrate having
a planar capillary holding surface, a plurality of capillaries
aligned on the capillary holding surface of the substrate,
scattered light shielding means placed on the plurality of
capillaries for shielding areas at which adjacent capillaries
contact with each other with a partial area including a central
axis of each of the capillaries being not shielded, wherein a laser
beam irradiated on a capillary at one end or capillaries at both
ends of the plurality of capillaries in a substantially parallel
direction with respect to the capillary holding surface propagates
to all the plurality of capillaries one by one to travel through
the capillaries, and emissions from each of the capillaries are
detected through the space between the shielding areas in a
substantially perpendicular direction with respect to the capillary
holding surface, wherein each of the capillaries is provided with a
coating, but is not provided with the coating in an area for
receiving the laser beam irradiation thereon, and the scattered
light shielding means contacts with the area of the capillaries for
which the coating is not provided.
[0014] In order to bring the shielding means into direct contact
with the coating-removed area of the capillary, a shape of a
contact portion of the shielding means with the capillary may be so
formed as to mate the capillary having a step-like shape that is
formed when the coating is partially removed, or a width of the
coating-removed portion of the capillary may be made wider than
that of the scattered light shielding means.
[0015] In order to achieve the above object, the present invention
provides a capillary array photodetector comprising a reference
surface with which contacts with a capillary holding surface of a
capillary array holding substrate that holds a capillary array,
fixing means for fixing the capillary array holding substrate
brought into contact with the reference surface by pressing the
capillary array holding substrate from the rear surface thereof, a
laser light source, an irradiation optical system for setting a
part of a light path extending from the light source to be
substantially parallel to the capillary holding surface of the
capillary array holding substrate contacting with the reference
surface, and a photodetector system for detecting emissions, the
capillary array photodetector detecting emissions from each of the
capillaries, which emissions being caused by laser beam that is
irradiated from the irradiation optical system on a capillary at
one end or capillaries at both ends of the plurality of capillaries
aligned on the capillary holding surface of the capillary array
holding substrate fixed on the reference surface with being brought
into contact therewith and that propagates to all the plurality of
capillaries one by one to travel through the capillaries, wherein
the fixing means is provided with a recessed portion opening on a
side of the reference surface.
[0016] Since it is possible to secure a yet wider space in the rear
of the capillaries by attaching the capillary array of the present
invention wherein the substrate is provided with the perforation to
the capillary array photodetector for detection, opportunities for
light that will be reflected from the capillaries to enter the
photodetector are reduced, thereby further improving the effect of
reducing the crosstalk. In addition, in the case where a capillary
array holding means for improving handling of the capillary array
is provided in the rear of the capillaries when the capillary array
is viewed from the photodetector, the capillary array holding means
is also provided with a perforation in order to secure the space as
wide as possible in the rear of the capillaries when the capillary
array is viewed from the photodetector.
[0017] It is preferable to subject a wall that is a background of
the capillaries when the capillary array is viewed from the
photodetector, such as a surface of the recess on the fixing
portion, to the light-scattering prevention treatment or the light
reflection prevention treatment. These treatments may be, but not
limited to, a non-fluorescent black coating, blackening treatment
of a copper surface or patching of a light absorption material.
[0018] According to the invention, it is possible to largely reduce
the crosstalk otherwise detected from a capillary adjacent to a
capillary on which the laser beam is irradiated. In the case where
a detection limit of DNA sample is determined on the crosstalk from
the adjacent capillaries, the present invention can improve the
detection limit and increase the dynamic range.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a schematic view of an example of an
electrophoresis device according to the present invention;
[0020] FIG. 2A and FIG. 2B each is a schematic view of an example
of a capillary array according the present invention;
[0021] FIG. 2A is a front view of a part of the capillary array
fixed on a planar glass substrate, and
[0022] FIG. 2B is a sectional view taken along the line A-A of FIG.
2A;
[0023] FIG. 3A and FIG. 3B are block diagrams respectively showing
a detection unit of the capillary array and a guiding path of a
laser beam;
[0024] FIG. 3A is a schematic front view of an example of an
optical system of a capillary array photodetector according to the
present invention, and
[0025] FIG. 3B is a top view showing an example of a detection unit
of the capillary array;
[0026] FIG. 4 is a schematic diagram showing a laser beam path in a
cross-section of the capillary array;
[0027] FIG. 5 is a top view showing an example of a fluorescence
detection system;
[0028] FIG. 6 is a view showing an example of a method of mounting
the capillary array to the capillary array photodetector;
[0029] FIG. 7 is a graph showing measurement results of crosstalk
in the capillary array according to the present invention;
[0030] FIG. 8A, FIG. 8B and FIG. 8C each shows a state wherein a
capillary array is mounted on a glass substrate according to a
conventional system;
[0031] FIG. 8A is a front view,
[0032] FIG. 8B is a side view taken along the line A-A of FIG. 8A,
and
[0033] FIG. 8C is a sectional view viewed from a direction parallel
to a light path of a laser beam;
[0034] FIG. 9 is a graph showing measurement results of crosstalk
in a conventional capillary array.
[0035] FIG. 10 is a sectional view of another example of a
capillary array photodetector and a vicinity of an area on which
laser beam is irradiated according to the present invention;
[0036] FIG. 11 is a front view of FIG. 10;
[0037] FIG. 12A and FIG. 12B each is a schematic view of another
example of a capillary array according to the present
invention;
[0038] FIG. 12A is a front view of a part of the capillary array
fixed on a planar glass substrate, and
[0039] FIG. 12B is a sectional view taken along the line A-A of
FIG. 12A;
[0040] FIG. 13A and FIG. 13B each is a schematic view of another
example of a capillary array according to the present
invention;
[0041] FIG. 13A is a front view of a part of the capillary array
fixed on a planar glass substrate, and
[0042] FIG. 13B is a sectional view taken along the line A-A of
FIG. 13A;
[0043] FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D each shows a
capillary array with a mask;
[0044] FIG. 14A is a front view of the capillary array with a
mask,
[0045] FIG. 14B is a sectional view taken along the line A-A of a
conventional capillary array with a mask,
[0046] FIG. 14C is a sectional view taken along the line A-A of an
example of a capillary array according to the present invention,
and
[0047] FIG. 14D is a sectional view taken along the line B-B of the
capillary array according to the present invention; and
[0048] FIG. 15A and FIG. 15B each shows another example of a
capillary array with a mask according to the present invention;
[0049] FIG. 15A is a front view thereof, and
[0050] FIG. 15B is a sectional view taken along the line A-A of
FIG. 15A.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] Embodiments of the present inventions will hereinafter be
described with reference to the attached drawings. Electrophoresis
using samples each containing DNA will be described below by way of
examples.
[0052] First Embodiment
[0053] FIG. 1 is a schematic view of an electrophoresis device
according to the present invention. At one end of a capillary array
1, an electrode (sample inlet end) 2 is formed so that it can apply
a negative voltage. The voltage is applied after soaking the
negative electrode 2 into a solution containing the sample DNA (not
shown) in the case of injecting the sample DNA, and after soaking
the negative electrode 2 into a buffer solution 3 in the case of
conducting electrophoresis of the injected sample DNA. Formed on
the other end of the capillary array 1 is a connecting portion 5
for connecting the capillary array 1 with a gel block 4 from which
an electrophoresis medium gel is injected into a capillary. In
order to fill the capillary with the electrophoresis medium gel, a
valve 6 is closed and a syringe 10 is pressed so that the gel
retained in the syringe 10 is injected into the capillary 1. In the
case of conducting the electrophoresis, the valve 6 is opened, and
a voltage is applied between the negative electrode 2 soaked into
the buffer 3 and an earth electrode 7 soaked into a buffer 9. The
DNA in the sample is separated into fragments according to
molecular weights and so forth as it migrates to the earth
electrode 7 from the negative electrode 2 in the capillary 1 to
generate a DNA band in the capillary 1. A fluorescent dye molecule
is bonded to each of the synthetic DNA fragments. Laser beam
irradiated on an irradiation area 8 of the capillary array 1 causes
the fluorescent dye molecules to emit light, so that emission
spectra of the emissions are measured by fluorescence measuring
means to determine a base sequence and a base length of the DNA.
Temperature in the capillary 1 is maintained constant by a
gas-circulation-type oven 11. It should be noted that, in this
specification, a section in the capillary array electrophoresis
device shown in FIG. 1, which relates to the fluorescence
measurement, is sometimes referred to as a capillary array
photodetector.
[0054] Referring to FIG. 2A and FIG. 2B, an example of the
capillary array of the present invention will be described. Each of
these drawings shows a structure of a glass substrate and a state
wherein the capillary array is fixed on the glass substrate. FIG.
2A is a front view of a part of the capillary array fixed on the
planar grass substrate, and FIG. 2B is a sectional view taken along
the line A-A of FIG. 2A.
[0055] In the examples shown in the drawings, the capillary array
is formed of sixteen capillaries 21 that are aligned on a capillary
holding surface 75 as a planar surface of a flat grass substrate 20
and fixed thereon with an adhesive or the like. Each of the
capillaries 21 is a silica tube with a polymer coating. A portion
to be irradiated with a laser beam, which will be described later
in this specification, is not formed with the polymer coating with
the silica tube being exposed. Inner and outer diameters of the
silica tube respectively are 50 .mu.m and 323 .mu.m, and an outer
diameter of the capillary including the polymer coating is 363
.mu.m. Pitch of the capillaries is 363 .mu.m, which is equal to the
capillary outer diameter, and a width of the array is 5.8 mm (363
.mu.m.times.16).
[0056] The planar glass substrate 20, on which the capillaries 21
are aligned is formed with a perforation 72 at an area in the rear
of the capillaries when the capillary array is viewed from a
fluorescence detector, which will be described later in this
specification. On the surface of the glass substrate 20 excluding
the perforation 72, grooves 73 are formed along light paths for
laser beams in order to prevent the laser beams from contacting the
surface of the substrate.
[0057] FIG. 3A and FIG. 3B are block diagrams respectively showing
a detection unit of the capillary array and a guiding path for
laser beam; FIG. 3A is a schematic front view of an example of an
optical system of a capillary array photodetector according to the
present invention, and FIG. 3B is a top view showing the detection
unit of the capillary array. A shutter, a filter and so forth are
provided in an optical system actually used, although they are not
shown in the drawings for brevity. In FIG. 3A, reference numeral 22
denotes a first capillary, and reference numeral 23 denotes a
sixteenth capillary.
[0058] A laser beam emitted by a laser 29 is split into halves by a
beam splitter 30, and two laser beams 25 and 26 thus obtained are
reflected from a reflection mirror 31 to enter the capillary array
respectively from two directions that are opposite to each other.
The laser beams 25 and 26 respectively are condensed by laser
condenser lenses 27 and 28 (f=50 mm), and then irradiated on the
fluorescence detection portion (laser irradiation portion) 24 of
the capillary array from both side surfaces thereof. Each of
distances between the laser condenser lens 27 and the first
capillary 22 and between the laser condenser lens 28 and the
sixteenth capillary 23 is 50 mm.
[0059] As shown in FIG. 4, the laser beam 25 guided into the first
capillary 22 propagates across the sixteen capillaries one by one
in such a manner that the laser beam 25 passes through the gel in
each of the capillaries while being refracted at an interface
between an ambient atmosphere and each of the silica tubes and at
an interface between each of the silica tubes and the gel inside
the silica tube and repeating focusing and divergence, thereby
exciting the fluorescent dye in the capillaries. Reference numeral
130 in the drawing denotes a polyimide coating peeled off for the
laser irradiation portion 24.
[0060] FIG. 5 is a top view showing schematically a fluorescence
detection system. An optical axis of each of the laser beams 25 and
26 is perpendicular to the drawing sheet. Emissions 40 in one of
the capillaries 21 are modified to be substantially parallel light
by using an emission condenser lens 41 (f=1.4) to be guided into a
transmission grating 42. The transmission grating 42 splits the
light to generate pieces of light 43 and 44 that are focused on a
two-dimensional CCD 46 via a focusing lens 45. Wavelength
dispersion direction according to the transmission grading 42 is
perpendicular to the laser optical axis. Therefore, one of the two
orthogonal axes on the two-dimensional CCD 46 represents space
coordinates of the direction of alignment of the sixteen
capillaries, and the other represents emission spectra from the
capillaries. From the emission spectra, fluorescence labeled DNA is
detected and identified.
[0061] FIG. 6 shows a method of mounting the capillary array to the
capillary array photodetector. In the multifocus irradiation
system, it is necessary to mount a reproducible capillary array
since a relative alignment precision between the laser optical axis
of the capillary array photodetector and the capillary array is
important. Therefore, as shown in FIG. 6, a part of the planar
glass substrate 20 on which the capillaries 21 are aligned is
brought into contact with a mounting reference surface 52 of a
capillary array photodetector 55, so that the capillary array is
pressed against the photodetector 55 by array fixing means 53. In
the example shown in FIG. 6, a capillary array holder 54 is so
mounted on the planar glass substrate 20 that handling of the
capillary array can be facilitated, and the array fixing means 53
presses the planar glass substrate 20 against the mounting
reference surface 52 of the capillary array photodetector 55 via
the capillary array holder 54.
[0062] The laser beams 25 and 26 propagate across the gel portions
of the sixteen capillaries one by one to excite the fluorescence
dyes bonded to the DNA migrating in the gel as described with
reference to FIGS. 3A to 5 in the state where a part of a capillary
fixing side plane of the planar glass substrate 20, on which the
capillary array is fixed, is contacted with and fixed to the
mounting reference surface 52 on the capillary array photodetector
55. The emissions from the fluorescent dyes are detected by the
fluorescence detection system in the capillary array photodetector
55 as described with reference to FIG. 5.
[0063] FIG. 7 is a graph showing measurement results obtained by
injecting a sample containing DNAs of a single base length into the
tenth capillary of the capillary array of the present invention. In
FIG. 7, fluorescent signals detected from a position of a ninth
capillary that is adjacent to the tenth capillary are plotted in
addition to fluorescent signals detected from a position of the
tenth capillary. It should be noted that values for the vertical
axis of the fluorescent signals detected from the ninth capillary
position are plotted as being magnified 100 times. Since the DNA
sample was injected only into the tenth capillary, the signals
observed at the ninth capillary position are crosstalk. The
crosstalk observed under these electrophoresis conditions was
0.1%.
[0064] In turn, FIG. 8A, FIG. 8B and FIG. 8C each shows a state
wherein a capillary array is mounted on a glass substrate according
to a conventional system; FIG. 8A is a front view, FIG. 8B is a
side elevation taken along the line A-A of FIG. 8A, and FIG. 8C is
a sectional view viewed from a direction parallel to a light path
of laser beam.
[0065] Sixteen capillaries 71 are aligned on a planar glass
substrate 70. On a capillary holding surface of the planar glass
substrate, a groove 62 is formed along a light path for laser beams
in order to avoid contact of laser beams with the planar glass
substrate 70. Due to the grooving, a glass surface 63 corresponding
to a part of the groove 62 is in the state of a frosted glass.
[0066] FIG. 9 is a graph showing measurement results when a sample
containing DNAs of a single base length is injected into the tenth
capillary in the conventional capillary array. In FIG. 9,
fluorescent signals detected from a position of a ninth capillary
that is adjacent to the tenth capillary are plotted in addition to
fluorescent signals detected from a position of the tenth
capillary. It should be noted that values for the vertical axis of
the fluorescent signals detected from the ninth capillary position
are plotted as being magnified 100 times. Since the DNA sample was
injected only into the tenth capillary, the fluorescent signals are
supposed to be detected only from the tenth capillary position, but
not from the ninth capillary position. However, as shown in FIG. 9,
there occurred crosstalk in the signals detected from the tenth
capillary position, which were also observed in the ninth capillary
position. In this case an amount of the crosstalk was 0.4%.
[0067] Thus, as it is apparent from the comparison with the
conventional system, according to the present embodiment, the
crosstalk was reduced owing to the perforation 72 formed in the
planar glass substrate in place of the conventional groove 62.
[0068] Second Embodiment
[0069] A second embodiment of the present invention will be
described with reference to FIGS. 10 and 11. FIG. 10, which
corresponds to FIG. 6, is a sectional view of another example of a
capillary array photodetector and a vicinity of an area on which
laser beam is irradiated. FIG. 11 is a front view of FIG. 10.
[0070] In the present embodiment, as shown in FIGS. 10 and 11, a
capillary array holder 81 placed at the rear of the planar glass
substrate 20 is formed with a perforation 80, in addition to the
perforation 72 formed on the planar glass substrate when the
capillary array is viewed from the fluorescence detector provided
in the capillary array photodetector. Further, a cavity 84 is
formed on a side facing to the capillary array of an array fixing
means 82 for fixing the capillary array, so that emissions from the
capillaries 71 enter the cavity 84. A black coating that is
remarkably low in reflectance is applied on an inner wall 83 of the
cavity 84 provided on the array fixing means 82. Other
configuration are the same as those of the first embodiment.
[0071] According to the present embodiment, crosstalk detected with
respect to a capillary adjacent to a capillary on which the laser
beam was irradiated was 0.05% and, thus, it was confirmed that the
capillary array of the present invention reduces the crosstalk more
effectively than that achieved by the conventional capillary
array.
[0072] Third Embodiment
[0073] A third embodiment will be described with reference to FIGS.
12A and 12B. FIG. 12A is a front view of a part of a capillary
array fixed on a planar glass substrate, and FIG. 12B is a
sectional view taken along the line A-A of FIG. 12A.
[0074] Conventionally, the groove 62 has been formed along the
light path of laser beams on the planar glass substrate 70 in order
to avoid contact of the laser beam with the planar glass substrate
70 as described with reference to FIG. 8. The glass surface 63
corresponding to a part of the groove has been in the state of a
frosted glass due to the grooving. In turn, in the present
embodiment, grooves 90 for avoiding contact of the laser beams with
the planar glass substrate 91 are respectively formed on opposite
ends of a planar glass substrate 91, but not in an area in which
capillaries 92 are aligned.
[0075] There will be explained reasons for the sufficient avoidance
of contact of the laser beams with the planar glass substrate 91
that is achieved by the grooves formed at opposite ends of the
planar glass substrate according to the present embodiment. As
described with reference to FIGS. 3A and 3B, since the laser beams
are condensed by the lenses to be irradiated on the capillary
array, a diameter of each of the beams is increased as they
approach to the ends of the planar glass substrate 91, while it is
reduced as they approach to the capillaries 92. Since each of the
beam diameters is sufficiently smaller than a capillary diameter in
the vicinity of the capillaries 91 and each of the laser beams
enters each of the capillaries from an approximately center
position (a portion filled with the gel), the laser beams do not
contact with the planar glass substrate 91 though there is no
groove in the vicinity of the capillaries. However, at the ends of
the planar glass substrate 91, the laser beams possibly contact
with the planar glass substrate 91 if the grooves 90 are not
formed.
[0076] According to the present embodiment, crosstalk detected with
respect to a capillary adjacent to a capillary on which the laser
beam was irradiated was 0.25% and, thus, it was confirmed that the
capillary array of the present embodiment reduces the crosstalk as
compared with the conventional one.
[0077] Fourth Embodiment
[0078] A fourth embodiment will be described with reference to
FIGS. 13A and 13B. FIG. 13A is a front view of a part of the
capillary array fixed on a planar glass substrate, and FIG. 13B is
a sectional view taken along the line A-A of FIG. 13A.
[0079] A planar glass substrate 101 for fixing a capillary array of
the present embodiment is not provided with a groove for avoiding
contact of laser beams with the planar glass substrate. By
downsizing the planar glass substrate 101, or by irradiating the
planar glass substrate 101 with each of the laser beams at a
certain angle as indicated by a broken line 100 in FIG. 10B with
respect to the planar glass substrate 101, it is possible to avoid
contact of the laser beams with the glass pate 101.
[0080] As described below, it is possible to irradiate all the
capillaries of the capillary array with a laser beam by irradiating
the planar glass substrate with the laser beam at a certain angle
with respect, and the laser beam is not necessarily irradiated in
parallel with respect to the planar glass substrate. Since each of
the capillaries functions as a rod lens, it is possible to control
a laser beam outgoing angle, at which the laser beam outgoes to an
adjacent capillary from a capillary (end capillary) on which the
laser beam is firstly irradiated, depending on a laser beam
irradiation position of the end capillary. Therefore, by properly
setting the laser beam irradiation position of the first capillary,
it is possible to propagate the laser beam to all the capillaries
aligned in parallel to the planar glass substrate even if the laser
beam is irradiated on the capillary at a angle with respect to the
capillary array.
[0081] According to the present embodiment, crosstalk detected with
respect a capillary adjacent to a capillary on which the laser beam
was irradiated was 0.25% and, thus, it was confirmed that the
capillary array of the present embodiment reduces the crosstalk as
compared with the conventional one.
[0082] Fifth Embodiment
[0083] In the same configuration as that shown in FIG. 13, a
monolayer anti-reflection coating of magnesium fluoride (MgF.sub.2)
is formed on a glass substrate surface on which capillary array is
provided. Crosstalk detected in this embodiment was 0.2% and, thus,
it was confirmed that the capillary array of the present embodiment
reduces the crosstalk as compared with the conventional one.
[0084] Sixth Embodiment
[0085] A capillary array with a mask according to the present
embodiment will be described with reference to FIGS. 14A to 14D.
FIG. 14A is a front view of the capillary array with mask, FIG. 14B
is a sectional view taken along the line A-A of a conventional
capillary array with mask, FIG. 14C is a sectional view taken along
the line A-A of an example of a capillary array according to the
present invention, and FIG. 14D is a sectional view taken along the
line B-B of the capillary array according to the present
invention.
[0086] In the capillary array of the present embodiment, scattered
light is shielded by attaching a mask 110 to the capillary array
fixed on a planar glass substrate 113. The attachment of a mask to
a capillary array has been realized in the art. However, such
conventional mask as denoted by reference numeral 114 in FIG. 14B
contacts with a polyimide coating 111 of the capillary, but not
with a glass portion 112 of a capillary a coating of which is not
applied. Therefore, there is a gap between the glass 112 of the
capillary and the mask 114, through which the scattered light
passes from an capillary adjacent to a target capillary on which
the laser beam is irradiated to be detected as signals from the
target capillary, thereby causing crosstalk. Hence, in the present
embodiment, as shown in the sectional view of FIG. 14C and the
sectional view of FIG. 14D, a structure of the mask 114 which does
not cause a gap between the glass portion 112 of each of the
capillaries and the mask 110 is employed. That is to say, a
thickness of a portion of the mask 110 in an area covering the
irradiation portion provided with a window 115 for each capillary,
is increased so that the mask contacts with the glass portion 112
exposed by removing the coating on the capillary. An area between
the mask 110 and the coating-removed portion of the capillary may
be filled with a light shielding material.
[0087] According to the capillary array employing the mask
structure of the present embodiment, crosstalk detected with
respect to the capillary adjacent to the target capillary was 0.3%
and, thus, it was confirmed that the capillary array of the present
embodiment reduces the crosstalk as compared with the conventional
one.
[0088] Seventh Embodiment
[0089] Seventh embodiment will be described with reference to FIGS.
15A and 15B. FIG. 15A is a front view thereof, and FIG. 15B is a
sectional view taken along the line A-A of FIG. 15A.
[0090] In the capillary array of the present embodiment, scattered
light is shielded by attaching a mask 121 to the capillary array
fixed on a planar glass substrate 124. In the present embodiment, a
width of a coating-removed portion 120 of each of the capillaries
122 is made wider than a width of the mask 121, so that the mask
121 does not contact directly with the coating-removed portion 120
of each of the capillaries 122. In the present embodiment, too, no
gap exists between each of the capillaries and the mask.
[0091] According to the capillary array employing the mask
configuration of the present embodiment, crosstalk detected with
respect to a capillary adjacent to a capillary on which the laser
beam was irradiated was 0.3% and, thus, it was confirmed that the
capillary array of the present embodiment reduces the crosstalk as
compared with the conventional one.
* * * * *