1 SP306720WO00
DESCRIPTION
CELL NUCLEUS OBSERVATION SUBSTRATE AND CELL NUCLEUS
OBSERVATION APPARATUS
5 Technical Field
[0001] The present invention relates to a cell
nucleus observation substrate and a cell nucleus
observation apparatus. More particularly, the present
invention relates to a cell nucleus observation
10 substrate for fluorescence staining and morphological
observing cell nuclei.
Background Art
[0002] In the related art, cell nuclei of cells,
15 microorganisms and the like have been observed using a
microscope and the like, and types and properties of
the cells and the microorganisms have been determined
based on the morphology of the nuclei. For ease of the
morphological observation of the nuclei, a pigment for
20 staining the nuclei is used to stain the cells, the
microorganisms and the like prior to the observation.
[0003] For example, Non-Patent Documents 1 and 2
describe a technology that erythrocytes are stained by
Giemsa to observe the cells infected by malaria
25 parasite. In the apparatuses described in Non-Patent
Documents 1 and 2, erythrocytes infected by malaria
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parasite in blood are collected using a magnet, whereby
malaria diagnosis can be done conveniently and speedy.
[0004] As the pigment for staining the nuclei,
methylene blue that is a blue pigment used for the
5 above-mentioned Giemsa stain, hematoxylin that is a
blue purple pigment and the like have been used from
the past, for example. In recent years, fluorescent
pigments bonding to nucleic acids are used to stain the
nuclei. Examples of the fluorescent pigments include
10 Hoechst and DAPI.
[0005] In relation to the present invention,
conventionally known fluorescence as autofluorescence
shown by cells upon a fluorescence observation will be
described. One of the fluorescence is orange-colored
15 autofluorescence shown by UV-irradiated cells in the
presence of copper. For example, it is reported that
cells of a particular part of a drosophila larvae
midgut emits orange-colored fluorescence when copper is
added (see Non-Patent Documents 3 to 11). The cells
20 where the orange-colored fluorescence is especially
strongly observed in the drosophila larvae midgut are
called as "copper cells" or the like. It is reported
that the fluorescence is observed at cells around the
copper cells (Non-Patent Document 6) and an entire body
25 wall of the larvae (Non-Patent Document 4) when the
concentration of the copper added is increased.
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[0006] There is a description that the abovementioned
orange-colored fluorescence is observed at
both cytoplasms and cell nuclei in cells, and, in
particular, is detected predominantly in grains of the
5 cytoplasms (see Non-Patent Documents 4 to 6 and 9).
There is a description that a wavelength range of
fluorescence is 590 to 630 ran, a peak wavelength is 610
nm and a maximum excitation wavelength is 340 nm (see
Non-Patent Document 5).
10 [0007] Also for organisms other than drosophila,
autofluorescence having similar properties is observed.
For example, there is reported that orange-colored
fluorescence (having a peak wavelength of 605 nm) is
observed in an individual liver to which copper is
15 added by UV excitation (excitation wavelength of 310
nm) in rat experiments (see Non-Patent Document 11).
Furthermore, there is reported that similar
fluorescence is observed in a kidney of a model rat
having a kidney and a liver where copper is accumulated
20 with aging (see Non-Patent Document 12). Also, the
autofluorescence having similar properties is reported
in yeast (see Non-Patent Document 13) and human liver
tissues of a Wilson's disease patient (see Non-Patent
Document 14). The Wilson's disease is a genetic
25 disorder of insufficient excretion of copper and
accumulation of copper in liver cells.
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[0008] As the above-described fluorescent substance
emitting orange-colored fluorescence, a composite of
copper and metallothionein (MT) (hereinafter
abbreviated to as "Cu-MT") is presumed (see Non-Patent
5 Documents 16 to 25). The Cu-MT has wavelength
properties such as an excitation wavelength of 305 nm
and a fluorescence wavelength of 565 nm in Non-Patent
Document 15, and an excitation wavelength of 310 nm and
a fluorescence wavelength of 570 nm in Non-Patent
10 Document 19. It is conceivable that the Cu-MT contain
monovalent copper ions (Cu(I))(see Non-Patent Documents
15, 17, 19, 21 and 25).
[0009] As the fluorescent substance containing
copper, a compound containing pyrimidine or mercaptide
15 that emits fluorescence by interacting pyrimidine or
mercaptide with copper is widely known (see Non-Patent
Documents 26 to 31) .
[0010] On the other hand, an interaction of various
metal ions with nucleic acids has been traditionally
20 studied. For example, it is known that when monovalent
copper ions are interacted with nucleic acids, a minor
amount of copper contained in cell nuclei stabilizes a
nucleic acid structure, but hurts DNAs under
coexistence of hydrogen peroxide (see Non-Patent
25 Document 32) . It is also reported that an interaction
with copper changes an absorption spectrum of DNAs (see
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Non-Patent Documents 32 and 33) . Further, it is
reported that the change in the absorption spectrum
depends on base sequences (specifically, a polymer
having a G-C pair and a polymer having an A-T pair) of
5 the DNAs (see Non-Patent Document 32).
[0011]
Non-Patent Document 1: "Diagnosis of malaria by
magnetic deposition microscopy." Am. J. Trop. Med. Hyg.,
2006, Vol.74, No.4, p. 568-572
10 Non-Patent Document 2: "Enhanced detection of
gametocytes by magnetic deposition microscopy predicts
higher potential for Plasmodium falciparum
transmission." Malaria Journal, 2008, 7, 66
Non-Patent Document 3: Physiological genetic
15 studies on copper metabolism in the genus Drosophila.
(1950) Genetics 35, 684-685
Non-Patent Document 4: Organization and function
of the inorganic constituents of nuclei. (1952) Exp.
Cell Res., Suppl. 2:161-179
20 Non-Patent Document 5: Ultrastructure of the
copper- accumulating region of the Drosophila larval
midgut. (1971) Tissue Cell. 3, 77-102
Non-Patent Document 6: Specification of a single
cell type by a Drosophila homeoticgene. (1994) Cell. 76,
25 689-702
Non-Patent Document 7: Two different thresholds
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of wingless signalling with distinct developmental ]
consequences in the Drosophila midgut. (1995) EMBO J.
14, 5016-5026.
Non-Patent Document 8: Calcium-activated
5 potassium channel gene expression in the midgut of
Drosophila. (1997) Comp. Biochem. Physiol. B Biochem.
Mol. Biol. 118, 411-420
Non-Patent Document 9: Evidence that a coppermetallothionein
complex is responsible for fluorescence
10 in acid-secreting cells of the Drosophila stomach.
(2001) Cell Tissue Res. 304, 383-389
Non-Patent Document 10: Peptidergic paracrine and
endocrine cells in the midgut ofthe fruit fly maggot.
(2009) Cell Tissue Res. 336, 309-323
15 Non-Patent Document 11: A luminescence probe for
metallothionein in liver tissue:emission intensity
measured directly from copper metallothionein induced
in ratliver. (1989) FEBS Lett. 257, 283-286
Non-Patent Document 12: Direct visualization of
20 copper- metallothionein in LEC ratkidneys: application
of autofluorescence signal of copper-thiolate cluster.
(1996) J. Histochem. Cytochem. 44, 865-873
Non-Patent Document 13: Incorporation of copper
into the yeast Saccharomyces cerevisiae. Identification
25 of Cu(I)—metallothionein in intact yeast cells. (1997)
J.Inorg. Biochem. 66, 231-240
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Non-Patent Document 14: Portmann B. Image of the
month. Copper- metallothionein autofluorescence. (2009)
Hepatology. 50, 1312-1313
Non-Patent Document 15: Luminescence properties
5 of Neurospora copper metallothionein. (1981) FEBS Lett.
127, 201-203
Non-Patent Document 16: Copper transfer between
Neurospora copper metallothioneinand type 3 copper
apoproteins. (1982) FEBS Lett.142, 219-222
10 Non-Patent Document 17: Spectroscopic studies on
Neurospora copper metallothionein. (1983) Biochemistry.
22, 2043-2048
Non-Patent Document 18: Metal substitution of
Neurospora copper metallothionein. (1984) Biochemistry.
15 23, 3422-3427
Non-Patent Document 19: (Cu,Zn)-metallothioneins
from fetal bovine liver. Chemicaland spectroscopic
properties. (1985) J. Biol. Chem. 260, 10032-10038
Non-Patent Document 20: Primary structure and
20 spectroscopic studies of Neurosporacopper
metallothionein. (1986) Environ. Health Perspect. 65,
21-27
Non-Patent Document 21: Characterization of the
copper-thiolate cluster in yeast metallothionein and
25 two truncated mutants. (1988) J. Biol. Chem. 263, 6688-
6694
i
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Non-Patent Document 22: Luminescence emission
from Neurospora copper metallothionein. Time-resolved
studies. (1989) Biochem J. 260, 189-193
Non-Patent Document 23: Establishment of the
5 metal-to-cysteine connectivities in silver-substituted
yeast metallothionein (1991) J. Am. Chem. Soc. 113,
9354-9358
Non-Patent Document 24: Copper- and silversubstituted
yeast metallothioneins: Sequential proton
10 NMR assignments reflecting conformational heterogeneity
at the Cterminus. (1993) Biochemistry. 32, 6773-6787
Non-Patent Document 25: Luminescence decay from
copper(I) complexes of metallothionein. (1998) Inorg.
Chim. Acta. 153, 115-118
15 Non-Patent Document 26: Solution Luminescence of
Metal Complexes. (1970) Appl. Spectrosc. 24, 319 - 326
Non-Patent Document 27: Fluorescence of Cu, Au
and Ag mercaptides. (1971) Photochem. Photobiol. 13,
279-281
20 Non-Patent Document 28: Luminescence of the
copper—carbon monoxide complex of Neurospora
tyrosinase. (1980) FEBS Lett. Ill, 232-234
Non-Patent Document 29: Luminescence of carbon
monoxide hemocyanins. (1980) Proc.Natl. Acad. Sci.
25 U.S.A. 77, 2387-2389
Non-Patent Document 30: Photophysical properties
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of hexanuclear copper(I) and silver(I) clusters. (1992)
Inorg. Chem., 31, 1941-1945
Non-Patent Document 31: Photochemical and
photophysical properties of tetranuclearand hexanuclear
5 clusters of metals with dlO and s2 electronic
configurations. (1993) Ace. Chem. Res. 26, 220-226
Non-Patent Document 32: Interaction of copper(I)
with nucleic acids. (1990) Int. J. Radiat. Biol. 58,
215- 234
10 Non-Patent Document 33: Copper(I)-Catalyzed
Regioselective "Ligation" of Azidesand Terminal
Alkynes. (2002) Ang. Chem. Int. Ed. 41, 2596-2599
Summary of Invention
15 Problem to be solved by the Invention
[0012] In a method of observing cell nuclei by
nuclear staining using a conventional pigment, it is
required to fix cells, microorganisms and the like,
repeat immersion to a pigment solution and cleaning,
20 drying and so on. Therefore, it requires much effort
and time. Working proficiencies are required.
[0013] A main object of the present invention is to
provide a technology of staining nuclei of cells,
microorganisms and the like by a simple operation and
25 observing their forms.
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Means for solving the Problem
[0014]
The present inventors have discovered that nucleic
acids that are contacted with copper will emit
5 fluorescence by irradiating with light in ultraviolet
wavelength region, and have devised a cell nucleus
observation substrate and the like according to the
present invention by applying the discovery.
In other words, in order to solve the problem, the
10 present invention provides a cell nucleus observation
substrate including an introduction part into which a
sample liquid containing cells is introduced, and an
observation area within which the cells in the sample
liquid introduced from the introduction part is held,
15 copper being disposed on a flow path of the sample
liquid in the introduction part and the observation
area so that the copper being capable of contacting
with the sample liquid.
In the cell nucleus observation substrate, when
20 the cell in the sample liquid introduced from the
introduction part is contacted with copper, a nucleic
acid therein will emit fluorescence and is held within
the observation area. Accordingly, in the cell nucleus
observation substrate, the cell including the nucleus
25 being fluorescence stained can be observed at the
observation area.
j
11 SP306720WO00 |
In the cell nucleus observation substrate, the
copper is preferably formed and disposed on the flow
path. |
The cell nucleus observation substrate may include
5 a magnet that is disposed attachably and detachably and
forms a magnetic field within the observation area, and
the cells in the sample liquid flowing through the flow
path are held within the observation area based on a
magnetic force. In addition, in the cell nucleus
10 observation substrate, the observation area is disposed
in a space between two opposing substrates at a
predetermined distance. The cell nucleus observation
substrate includes an introduction part where the
sample liquid is introduced into the space, and an
15 absorption member that absorbs the sample liquid
introduced from the introduction part to the space, in
which the magnet is attached at a position between the
introduction part and the absorption member.
[0015] Furthermore, the present invention provides a
20 cell nucleus observation apparatus including an
introduction part into which a sample liquid containing
cells is introduced, an observation area within which
the cells in the sample liquid introduced from the
introduction part are held, a substrate where copper is
25 disposed on a flow path of the sample liquid in the
introduction part and the observation area so that the
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copper being capable of contacting with the sample
liquid, and an optical detecting means for irradiating
a light to the observation area and detecting
fluorescence generated.
5 In the cell nucleus observation apparatus, when
the cell in the sample liquid introduced from the
introduction part is contacted with copper, a nucleic
acid therein will emit fluorescence and is held within
the observation area. Accordingly, in the cell nucleus
10 observation apparatus, the observation area is
irradiated with the light, and the fluorescence
generated is detected, whereby a nuclear fluorescence
staining image of the cells can be observed.
In the cell nucleus observation apparatus, a
15 wavelength of the light irradiated by the optical
detecting means is 300 to 420 nm.
Effect of the Invention
[0016]
20 The present invention provides a technology of
staining nuclei of cells, microorganisms and the like
by a simple operation and observing their forms.
Brief Description of Drawings
25 [0017]
[Fig. 1] A schematic diagram of illustrating a
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configuration of a cell nucleus observation substrate
according to a first embodiment of the present
invention.
[Fig. 2] A schematic diagram of illustrating a
5 configuration of a cell nucleus observation substrate
according to a second embodiment of the present
invention.
[Fig. 3] Graphs each substituting a drawing and showing
a fluorescent spectrum and an RFU value obtained by
10 bringing ssDNAs into contact with CUSO4 having a varied
concentration under the condition of an S.A.
concentration of 50 mM; (A) shows the fluorescent
spectrum and (B) shows a peak RFU value (Example 1).
[Fig. 4] Graphs each substituting a drawing and
15 showing fluorescent spectra and an RFU value obtained
by bringing ssDNAs into contact with CuS04 having a
varied concentration under the condition of an S.A.
concentration of 50 mM; (A) shows the fluorescent
spectra and (B) shows a peak RFU value (Example 1).
20 [Fig. 5] Graphs each substituting a drawing and
showing fluorescent spectra and RFU values obtained by
bringing oligo-DNAs into contact with CuS04 having a
concentration of 0.4 mM under the condition of an S.A.
concentration of 4 mM (Example 1).
25 [Fig. 6] Graphs each substituting a drawing and !
showing a fluorescent spectrum and an RFU value
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obtained by bringing oligo-DNAs into contact with CuS04
having a concentration of 0.4 mM under the condition of
an S.A. concentration of 4 mM (Example 1) .
[Fig. 7] Graphs each substituting a drawing and
5 showing a change with elapsed time of a fluorescent
spectrum and absorption spectra obtained in oligo-DNAs
T(20), T(6) and T(3) under the condition of a CuS04
concentration of 0.4 mM and an S.A. concentration of 4
mM (Example 1); the upper graphs each show the
10 fluorescent spectra with an ordinate axis of an RFU
value (absolute value), the middle graphs each show the
fluorescent spectra with an ordinate axis of an RFU
value (relative value) and the lower graphs each show
the absorption spectra.
15 [Fig. 8] Graphs each substituting a drawing and
showing a change with elapsed time of fluorescent
spectra and absorption spectra obtained in oligo-DNAs
T(20), T(6) and T(3) under the condition of a CuS04
concentration of 0.4 mM and an S.A. concentration of 4
20 mM (Example 1); (A) shows a change with elapsed time of
the peak RFU value, and (B) shows a change with elapsed
time at a wavelength of 34 6 nm.
[Fig. 9] Graphs each substituting a drawing and
showing a two-dimensional fluorescent spectrum acquired
25 in oligo-DNAs T(20), T(6) and T(3).
[Fig. 10] A graph substituting a drawing and showing
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excitation spectra (broken lines) and fluorescent
spectra (solid lines) obtained in oligo-DNAs T(20),
T(6) and T(3) (Example 1).
[Fig. 11] Graphs each substituting a drawing and
5 showing fluorescent spectra obtained in oligo-DNAs
including a three base length sequence by combining
adenine and thymine (Example 1).
[Fig. 12] Graphs each substituting a drawing and
showing a maximum RFU value (A) and a peak FRU
10 wavelength (B) of the fluorescent spectra obtained in
oligo-DNAs including a three base length sequence by
combining adenine and thymine (Example 1).
[Fig. 13] Graphs each substituting a drawing and
showing the fluorescent spectra obtained in oligo-DNAs
15 including sequences of SEQ ID NOS. 19 and 20 (Example
1) •
[Fig. 14] A graph substituting a drawing and showing
fluorescent spectra obtained by bringing a sample
containing ssDNAs into contact with solid copper
20 (Example 2).
[Fig. 15] A graph substituting a drawing and showing a
fluorescent spectrum obtained by bringing a sample
containing ssDNAs into contact with solid copper having
different concentrations (Example 2).
25 [Fig. 16] Graphs each substituting a drawing and
showing fluorescent spectra obtained by bringing a
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sample containing ssDNAs into contact with a reaction
solution containing a salt having different types or
concentrations (Example 2).
[Fig. 17] Graphs each substituting a drawing and
5 showing fluorescent spectra obtained by bringing a
sample containing ssDNAs (A) or RNAs (B) having
different concentrations with into contact solid copper
(Example 2).
[Fig. 18] Graphs each substituting a drawing and
10 showing fluorescent spectra obtained by bringing a
sample containing oligo-DNAs having different sequences
into contact with solid copper (Example 2).
[Fig. 19] Graphs each substituting a drawing and
showing a fluorescent spectrum obtained by bringing a
15 sample containing oligo-DNAs having different sequences
into contact with solid copper (Example 2).
[Fig. 20] Graphs each substituting a drawing and
showing excitation-fluorescent spectra obtained by
bringing a sample containing oligo-DNAs having
20 different sequences into contact with solid copper
(Example 2).
[Fig. 21] A graph substituting a drawing and showing
fluorescent spectra obtained in oligo-DNAs having
combination sequences of eight-base cytosine and 12-
25 base thymine (Example 2).
[Fig. 22] Graphs each substituting a drawing and
I ,
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showing fluorescent spectra obtained in double-stranded
DNAs including a mismatch (Example 2) .
[Fig. 23] Graphs each substituting a drawing and
showing RFU values obtained by changing a type and a pH
5 of a buffer of a reaction solution (Example 2).
[Fig. 24] Photographs each substituting a drawing and
showing a fluorescent image obtained by bringing copper
sputtered on a glass surface into contact with ssDNAs
(Example 3).
10 [Fig. 25] Photographs each substituting a drawing and
showing a fluorescent image obtained by bringing copper
sputtered on a glass surface into contact with RNAs
(Example 3).
[Fig. 26] A graph substituting a drawing and showing a
15 fluorescence intensity obtained by bringing copper or
silver sputtered on a glass surface into contact with a
sample including DNAs or RNAs (Example 3).
[Fig. 27] A graph substituting a drawing and showing a
change with elapsed time in a fluorescence intensity
20 obtained by bringing copper sputtered on a glass
surface into contact with ssDNAs (Example 3).
[Fig. 28] A graph substituting a drawing and showing a
change in a fluorescence intensity when a temperature
is changed after copper sputtered on a glass surface is
25 contacted with ssDNAs (Example 3).
[Fig. 2 9] Photographs each substituting a drawing and
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showing results of fluorescence observation of an onion
thin skin on a copper sputtered glass (Example 4).
[Fig. 30] Photographs each substituting a drawing and
showing results of fluorescence observation of a human
5 leukocyte sample on a copper sputtered glass (Example
4) .
[Fig. 31] Photographs each substituting a drawing and
showing results of fluorescence observation of Jurkat
cells on a copper sputtered glass (Example 4).
10 [Fig. 32] Photographs each substituting a drawing and
showing results of fluorescence observation of Jurkat
cells on a copper sputtered glass (Example 4).
[Fig. 33] Photographs each substituting a drawing and
showing magnetic labeled antibodies (EasySep-CD45)
15 labeled cells accumulated on the observation area of
the substrate by a magnet in Example 5.
[Fig. 34] Photographs each substituting a drawing and
showing magnetic labeled antibodies (MACS-CD45) labeled
cells accumulated on the observation area of the
20 substrate by a magnet in Example 5.
[Fig. 35] Photographs each substituting a drawing and
showing a permeated image (A) and a fluorescence image
(B) captured of magnetic labeled antibodies (MACS-CD45)
labeled cells accumulated on the observation area of
25 the substrate on which copper was sputtered in Example
5.
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Modes for Carrying Out the Invention
[0018] Hereinafter, embodiments according to the
present technology will be described with reference to
5 the drawings. The embodiments described below are
provided for purposes of illustration only, and merely
depict typical embodiments of the present technology,
and the scope of the present technology should not be
construed narrower. The embodiments will be described
10 in the following order.
1. Cell Nucleus Observation Substrate
(1) First Embodiment
(2) Second Embodiment
2. Cell Nucleus Observation Apparatus
15 [0019]
1. Cell Nucleus Observation Substrate
(1) First Embodiment
Fig. 1 is a schematic diagram of illustrating a
configuration of a cell nucleus observation substrate
20 according to a first embodiment of the present
invention. (A) is a top view, (B) is a sectional view
corresponding to a P-P section in (A), and (C) is a
sectional view corresponding to a Q-Q section in (A).
[0020] In the Figure, a cell nucleus observation
25 substrate designated in a symbol A includes a substrate
layer ai and a substrate layer a2. The substrate layer
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ai faces to the substrate layer a2 at a predetermined
space via a spacer a3. The space has a non-limiting
thickness, but the thickness is desirably from several
micrometers to hundreds micrometers, preferably about
5 10 to 50 micrometers. At a part of the space between
the substrate layer ai and the substrate layer a2, an
observation area 2 is disposed as an observation site
of cells.
[0021] A part of the space formed by the substrate
10 layer ai and the substrate layer a2 is closed by the
spacer a3, and the rest thereof is open and is
communicated with exterior. Specifically, upper and
lower two sides of the space formed by the substrate
layer ai and the substrate layer a2 is closed by the
15 spacer a3, and left and right two sides of the space is
communicated with exterior in Fig. (A) . A part of the
open space between the substrate layer ai and the
substrate layer a2 (in Fig. (A) , a left side) is an
introduction part 1 of a sample liquid to the
20 observation area 2 disposed at the space. At a part of
the open space (in Fig. (A), a right side) on the
opposite side of the introduction part 1, an absorption
member 4 that absorbs the sample liquid introduced to
the space from the introduction part 1 is inserted into
25 and disposed between the substrate layer ai and the
substrate layer a2. The sample liquid introduced from
#
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the introduction part 1 migrates the space between the
substrate layer ai and the substrate layer a2, and is
| absorbed by the absorption member 4, whereby the cells
contained in the sample liquid are held within the
5 observation area 2.
[0022] As the sample liquid, a buffer liquid
containing a salt such as sodium chloride (NaCl),
potassium chloride (KC1), magnesium chloride (MgCl2) is
preferably used (see Example 2 later). One salt or two
10 or more of salts may be contained, and the
concentration thereof is not especially limited.
Preferably, the concentration is set to 0.025 M or more.
The buffer for use in the sample liquid may not contain
a cheating agent for Cu(II) ions (see Example 2). For
15 the purpose of decreasing a damage to cells, the buffer
liquid that is adjusted to have a salt concentration
almost being isotonic to physiological tissues and
cells (for example, physiological saline) is desirably
used as the sample liquid. As the buffer liquid,
20 phosphoric buffer physiological saline (PBS) using
phosphoric buffer may be used (see Example 2).
[0023] The absorption member 4 is not especially
limited as long as the sample liquid can be absorbed,
and may be, for example, filter paper, absorption fiber
25 and absorption resin.
[0024] On the surface of the substrate layer a2
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facing to the substrate layer ai, a copper film 3 is
formed. The cells in the sample liquid introduced from
the introduction part 1 migrates the space between the
substrate layer ai and the substrate layer &2 while
5 contacting with the copper eluted into the sample
liquid. In this case, when nucleic acids are contacted
with copper, the nucleic acids in cell nuclei will emit
fluorescence by irradiating with light in ultraviolet
wavelength region.
10 [0025] The copper film 3 is formed by sputtering or
vapor deposition on a surface of the substrate using a
conventionally known method. A thickness of the copper
film 3 is not especially limited, but is several runs to
several tens runs, preferably about 20 to 40 ran. Within
15 the film thickness range, the copper film 3 has a light
permeability. It is therefore possible to irradiate a
light or detect fluorescence permeating through the
copper film 3, when the cells held within the
observation area 2 are irradiated with the light and a
20 nuclear fluorescence staining image of the cells is
observed. The light irradiation and the fluorescence
detection can be done at a surface opposite to the
copper film 3 using an incident-light optical system.
In this case, the copper film may not have light
25 permeability.
[0026] In the Figure, reference numeral 5 designates
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a magnet for forming a magnetic field within the
observation area 2. The magnet 5 is disposed on the
substrate layer ai adjacent to its surface opposite to
the observation area 2. The magnet 5 may be disposed
5 attachably and detachably at either side of the
substrate layer ax and the substrate layer a2. The
magnet 5 selects, concentrates and holds the cells in
the sample liquid, which migrate the space between the
substrate layer ai and the substrate layer a2, within
10 the observation area 2 based on a magnetic force of the
magnetic field formed within the observation area 2. In
this way, only the cells adsorbed to a magnetic
substance can be accumulated within the observation
area 2.
15 [0027] A material of the magnet 5 is not especially
limited as long as the magnetic field can be formed
within the observation area 2, and can be a neodymium
magnet, a samarium-cobalt magnet or a ferrite magnet,
for example. Also, a position of the magnet 5 is not
20 limited as long as the magnetic field can be formed
within the observation area 2, and may be buried into
the substrate layer ai or the substrate layer a2. In
the cell nucleus observation substrate according to the
present invention, the magnet 5 will not be an
25 essential constituent.
[0028] The cells accumulated within the observation
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area 2 are in the state that nucleic acids within the
cell nuclei emit fluorescence once they are in contact
with copper and are irradiated with ultraviolet rays.
Accordingly, the observation area 2 is irradiated with
5 ultraviolet rays and fluorescence generated is detected,
thereby observing the nuclear fluorescence staining
image of the cells.
[0029] A wavelength of the light to be irradiated is
preferably 300 to 420 ran. As the material of the
10 substrate layer ai and the substrate layer a2, a
material having light permeability, less
autofluorescence, and less optical error due to a small
wavelength distribution is preferably selected in order
to permeate the light to be irradiated to the
15 observation area 2 and fluorescence generated from the
cell nuclei. As the material of the substrate layer ai
and the substrate layer a2, glass and various plastics
(polypropylene, polycarbonate, cycloolefin polymer,
polydimethyl siloxane etc.) can be used. In addition,
20 in order that the light irradiated to the observation
area 2 and the fluorescence generated from the cell
nuclei are prevented from blocking by the magnet 5, the
magnet 5 disposed on the substrate layer ai can be
removed when the nuclear fluorescence staining image is
25 observed. Alternatively, using an incident-light
optical system, the light irradiation and the
25 SP306720WO00
fluorescence can be observed from the surface opposite
to the magnet.
[0030] As described above, in the cell nucleus
observation substrate A, the cells having magnetism in
5 the sample liquid introduced from the introduction part
1 are accumulated within the observation area 2 and are
contacted with the copper film 3 to be in the state
that nucleic acids within the cell nuclei emit
fluorescence, whereby the nuclear fluorescence staining
10 image of the cells can be observed. Accordingly, in the
cell nucleus observation substrate A, the cell nuclei
can be fluorescence stained by a simple operation and
can be observed their forms without requiring fixing
cells, microorganisms and the like, repetitive
15 immersion to a pigment solution and cleaning, drying
and so on that are required in the cell nucleus j
i
observation method by nuclear staining using
conventional pigments including the Giemsa stain.
[0031] In particular, in the cell nucleus
20 observation substrate A, as the cells can be
accumulated within the observation area 2 by the magnet
5, and can be observed, it is suitable for nucleus
observation of the cells having magnetism. Examples of
the cells having magnetism include cells labeled with
25 magnetic labeled antibodies and malaria parasite cells
that erythrocytes are infected as described in the
i
i
26 SP306720WO00
above-mentioned Non-patent documents 1 and 2.
[0032]
In the cell nucleus observation substrate A, when
the sample liquid containing the cells provided by
5 labeling a specific cell population with magnetic
labeled antibodies is used, only the specific cell
population can be accumulated within the observation
area 2 based on a magnetic action between the magnet 5
and the magnetic labeled antibodies. Accordingly, the
10 nuclei can be observed by concentrating only leucocytes
in a blood sample, which can be used for diagnosis of
leukemia based on a nucleus shape or of allergy based
on eosinophil counts. The specific cell population can
be labeled using the magnetic labeled antibodies
15 specifically bonded to specific cell surface antigens.
It is also possible that an antibody labeling area is
disposed between the introduction part 1 and the
observation area 2, and the magnetic labeled antibodies
are mixed with the cells in the antibody labeling area.
20 [0033] In addition, in erythrocytes infected by
malaria parasite, it is known that a magnetic substance
called Hemozoin is formed. In the cell nucleus
observation substrate A, when a blood sample collected
from a malaria patient is used, only the erythrocytes
25 infected by malaria parasite can be accumulated within
the observation area 2 based on the magnetic action
27 SP306720WO00 j
between the magnet 5 and Hemozoin. Accordingly, the
erythrocytes infected with the malaria parasite in the
blood sample can be concentrated to observe malaria
parasite nuclei. It is possible that malaria infection
5 and a malaria parasite type can be easily and
accurately determined. In this case, in the cell
nucleus observation substrate A, the nuclear
fluorescence staining image can be observed and
determined without requiring fixing, staining, drying
10 and so on different from malaria diagnosis by the
conventional Giemsa stain.
[0034]
As the malaria parasite, tropical malaria parasite,
tertian malaria parasite, quartan malaria parasite,
15 oval malaria parasite or the like is known. In addition,
as a growing stage of a parasite body, ringform,
trophozoite, schizont, gametocyte or the like is known.
It is very important to adequately identify the type
and the growing stage of the parasite for deciding a
20 therapeutic strategy of the infected patient.
[0035] In the cell nucleus observation substrate A
as described above, the case that the copper film 3 is
formed on the surface of the substrate layer a2 facing
to the substrate layer ai is described as an example.
25 In the cell nucleus observation substrate A, the copper
film 3 may be formed at any area of a flow path of the
28 SP306720WO00
sample liquid in the introduction part 1 and the
observation area 2 so that the copper film 3 can be in
contact with the sample liquid. For example, the copper
film 3 may be formed at a part of or entire surface of
5 the substrate layer aj.. In the cell nucleus observation
substrate according to the present invention, copper
may be disposed as not only a film but as powder, fine
particles, wire, plate and foil. In addition, in the
cell nucleus observation substrate according to the
10 present invention, copper is preferably a solid state
from a standpoint that the substrate is stably stored.
However, a copper solution may be used. When the copper
solution is used, it is preferable that a reducing
agent is mixed therewith in order to maintain the state
15 that dissolved Cu ions are reduced from divalent cation
to monovalent cation. As the reducing agent, sodium
ascorbate can be used as described later (see Example
1) . According to the present invention, the term
"copper" involves a copper metal and a copper alloy.
20 [0036]
(2) Second Embodiment
Fig. 2 is a schematic diagram of illustrating a
configuration of a cell nucleus observation substrate
according to a second embodiment of the present
25 invention. (A) is a top view, (B) is a sectional view
corresponding to a P-P section in (A).
29 SP306720WO00
[0037] In the Figure, a cell nucleus observation
substrate designated by a symbol B is configured by
adhering the substrate layer ax and the substrate layer
a2. In the substrate layer ai, the introduction part 1
5 into which the sample liquid is introduced and an
outlet 6 from which the sample liquid is discharged are
formed. On the substrate layer a2/ a flow path for
passing the sample liquid introduced from the
introduction part 1. At a part of the flow path, the
10 observation area 2 is disposed as a site for observing
the cells.
[0038] A liquid feeding means (not shown) is
connected to the introduction part 1 and the outlet 6.
The sample liquid introduced from the introduction part
15 1 passes through the flow path, and is discharged from
the outlet 6. The liquid feeding means is configured of
a general-purpose pump, tube, sample liquid tank, waste
tank or the like. In addition, the liquid feeding means
may include a configuration that the sample liquid is
20 introduced, fed and discharged utilizing centrifugal
force or gravity.
[0039] On a surface of the flow path formed on the
substrate layer a2, the copper film 3 is formed. The
copper film 3 may be fully formed over the flow path
25 surface. Alternatively, the copper film 3 may be formed
partly at the surface of the flow path of the sample
30 SP306720WO00
liquid in an upstream flow direction not at the
observation area 2, as shown in the Figure. Or, the
copper film 3 may be disposed on the substrate layer ai.
The cells in the sample liquid introduced from the
5 introduction part 1 to the flow path are contacted with
the copper film 3 or copper eluted from the copper film
3, and flow into the observation area 2. In this case,
the nucleic acids in the cell nuclei that are contacted
with copper will emit fluorescence by irradiating with
10 ultraviolet rays.
[0040] The copper film 3 is formed by sputtering or
vapor deposition on a surface of the substrate using a
conventionally known method. A thickness of the copper
i
film 3 is not especially limited, but is several nms to
15 several tens nms, preferably about 20 to 40 nm. Within
the thickness range, the copper film 3 has a light
i
permeability. It is therefore possible to irradiate a
light or detect fluorescence permeating through the
copper film 3, when the cells held within the
20 observation area 2 are irradiated with the light and a
nuclear fluorescence staining image of the cells are
observed.
[0041] In the Figure, reference numeral 5 designates
a magnet for forming a magnetic field within the
25 observation area 2. The magnet 5 is disposed attachably
and detachably on the substrate layer ax adjacent to its
31 SP306720WO00
surface opposite to the observation area 2. The magnet
5 holds the cells in the sample liquid, which are
introduced from the introduction part 1 and flow the
flow path, within the observation area 2 based on a
5 magnetic force of the magnetic field formed within the
observation area 2. In this way, by the magnet 5, the
cells that are contacted with copper and will emit
fluorescence are accumulated within the observation
area 2. As the material of the magnet 5 is similar to
10 that of the cell nucleus observation substrate A in the
first embodiment, a description thereof will be herein
omitted.
[0042] Also in the cell nucleus observation
substrate according to this present embodiment, the
15 magnet 5 will not be an essential constituent. As a
configuration to accumulate the cells within the
observation area 2, a substance for capturing the cells
in the sample liquid flowing (for example, an antibody
against a cell membrane antigen for capturing specific
20 cells) may be solid-phased, instead of the magnet 5.
Alternatively, an electrode for forming an electric
field within the observation area 2 is disposed instead
of the magnet 5, and the cells may be held within the
observation area 2 based on an electrical force.
25 [0043] The cells accumulated within the observation
area 2 are in the state that nucleic acids within the
32 SP306720WO00
cell nuclei emit fluorescence once they are in contact
with copper and are irradiated with ultraviolet rays.
Accordingly, the observation area 2 is irradiated with
ultraviolet rays and fluorescence generated is detected,
5 thereby observing the nuclear fluorescence staining
image of the cells.
[0044] A wavelength of the light to be irradiated is
preferably 300 to 420 nm. As the material of the
substrate layer ai and the substrate layer a2, a
10 material having light permeability, less
autofluorescence, and less optical error due to a small
wavelength distribution is preferably selected in order
to permeate the light to be irradiated to the
observation area 2 and fluorescence generated from the
15 light and the cell nuclei. As the material of the
substrate layer ai and the substrate layer a2, glass and
various plastics (polypropylene, polycarbonate,
cycloolefin polymer, polydimethyl siloxane etc.) can be
used. In addition, in order that the light irradiated
20 to the observation area 2 and the fluorescence
generated from the cell nuclei are prevented from
blocking by the magnet 5, the magnet 5 disposed on the
substrate layer ai can be removed when the nuclear
fluorescence staining image is observed. Alternatively,
25 using an incident-light optical system, the light
irradiation and the fluorescence can be observed from
33 SP306720WO00
the surface opposite to the magnet.
j
[0045] As described above, in the cell nucleus
observation substrate B, the cells in the sample liquid
introduced from the introduction part 1 are in contact
5 with the copper film 3 and accumulated within the
observation area 2 to be in the state that nucleic
acids within the cell nuclei emit fluorescence, whereby j
I
the nuclear fluorescence staining image of the cells I
can be observed. Accordingly, in the cell nucleus
10 observation substrate B, the cell nuclei can be
j
I
fluorescence stained by a simple operation and can be
observed their forms without requiring fixing cells,
microorganisms and the like, repetitive immersion to a
pigment solution and cleaning, drying and so on that
15 are required in the cell nucleus observation method by
nuclear staining using conventional pigments including
the Giemsa stain.
[0046]
2. Cell Nucleus Observation Apparatus
20 The cell nucleus observation apparatus according
to the present invention includes the above-described
cell nucleus observation substrate and an optical
detecting means for irradiating a light to the
observation area of the substrate, and thus can observe
25 the nuclear fluorescence staining image of the cells.
The cell nucleus observation apparatus may be provided
34 SP306720WO00
by changing a conventionally known fluorescence
microscope as appropriate, and includes the following
configuration as the optical detecting means.
[0047] The light to be irradiated to the observation
5 area has preferably within an ultraviolet region from
300 to 420 ran. As the light source, a laser, an LED or
the like can be used. Preferably, a UV-LED having a
central wavelength of about 360 nm is used. The light
from the light source is guided to the observation area
10 by an optical path configured of an optical filter, a
mirror, a lens or the like, as required.
[0048]
The fluorescence generated from the observation
area is detected by an optical detector such as a photo
15 detector, a photo diode, a photo multiplier, a CCD
camera, a CMOS camera and the like, and is displayed on
an image display apparatus. Alternatively, the
fluorescence generated from the observation area may be
observed with the naked eye via an ocular lens etc. The
20 fluorescence generated from the observation area is
guided by the optical path configured of the optical
filter, the mirror, the lens or the like, as required,
to the optical detector or the ocular lens. The optical
filter used has selectivity to the light having about
25 600 nm, which is in a fluorescence wavelength region,
generated from the nucleic acids contacted with copper
i
35 SP306720WO00
(see Example 1 below).
Example 1
[0049] Example 1 illustrates that orange-colored
fluorescence was emitted by ultraviolet irradiation
5 under certain conditions when nucleic acids are mixed
with a solution including Cu(I) ions generated by
reducing Cu(II) ions with an ascorbic acid.
[0050]

10 Cu: a CuS04 solution and (+)-Sodium L-ascorbate
(hereinafter referred to as "S.A.") were purchased from
Sigma-Aldrich.
Nucleic acid: Sonicated Salmon Sperm DNA
(hereinafter referred to as "ssDNA") purchased from
15 BioDynamics laboratory Inc. (Tokyo, Japan) was used. In
addition, as oligo-DNAs, Custom Oligo purchased from
Invitrogen Corporation was used.
Buffer: HEPPSO purchased from DOJINDO Laboratories
(Kumamoto, Japan) was used by adjusting the pH to 8.5
20 pursuant to the protocols provided by the manufacturer.
Fluorophotometer: NanoDrop 3300 (Thermo Fisher
Scientific, Inc., Waltham, MA, USA) or type F-4500
spectrofluorophotometer (Hitachi High-Technologies
Corporation) was used. In the NanoDrop 3300, a UV LED
25 light source was used to provide exciting light. A
fluorescent spectrum excited by the exciting light was
I
36 SP306720WO00
measured. Using a companion software, Relative
Fluorescence Units (RFU) at a wavelength where a
spectrum intensity became at maximum was acquired as a
peak RFU value. In the type F-4500
5 spectrofluorophotometer, a quartz capillary and a
dedicated adapter cell manufactured by Helix Biomedical
Accessories, Inc. were used. Unless otherwise noted
below, the NanoDrop 3300 was used.
Spectrophotometer: NanoDrop 1000
10 Spectrophotometer was used to measure an absorption
spectrum.
Sample preparation and fluorescence measurement:
50 mM of a HEPPSO buffer was mixed with sodium chloride
(250 mM), CuS04 (0 to 4 mM), S.A. (4, 50 mM), ssDNAs (1
15 mg/ml) or oligo-DNAs (50, 250, 500 uM) to provide 20 ul
of a sample. It is known that the S.A. has an action to
reduce Cu(II) ions generated from CUSO4 in the solution
to Cu(I) (see Non-Patent Document 33).
[0051]
20
Figs. 3 and 4 are graphs each showing a
fluorescent spectrum and an RFU value obtained by
changing a concentration of CuS04 under the condition of
an S.A. concentration of 50 mM; (A) shows the
25 fluorescent spectrum and (B) shows a peak RFU value.
[0052] Figs. 5 and 6 are graphs each showing
i
I
37 SP306720WO00
fluorescent spectra obtained under the conditions of
CUSO4 having a concentration of 0.4 mM and an S.A.
concentration of 4 mM. The oligo-DNAs having base
lengths of 20, 10, 6 and 3 had a concentration of 50,
5 50, 250, and 500 uM, respectively. Fig. 5 shows a
result of the oligo DNAs having the base sequences
described in SEQ ID NOS. 1 to 6. An abscissa axis
represents a wavelength, an ordinate axis in (A)
represents the RFU value in each wavelength, and an
10 ordinate axis in (B) represents a value provided by
dividing the RFU value in each wavelength by a maximum
RFU value. Fig. 6 shows a result (A) of the oligo-DNAs
having the base sequences described in SEQ ID NO. 2
(hereinafter described as T(20)), a result (B) of the
15 oligo-DNAs having the base sequences described in SEQ
ID NO. 10 (hereinafter described as T(6)), a result (C)
of the oligo-DNAs having the base sequences described
in SEQ ID NO. 12 (hereinafter described as T(3)), and a
result (D) of the oligo-DNAs having the base sequences
20 described in SEQ ID NO. 11 (hereinafter described as
T(3)). Each abscissa axis represents a wavelength, and
each ordinate axis represents an RFU value in each
wavelength.
[0053] As shown in the Figures, it was confirmed
25 that the patterns of the fluorescent spectra (a peak
wavelength and an intensity) were changed depending on
38 SP306720WO00
the base sequence's of the nucleic acids.
[0054] Next, a change with elapsed time of
fluorescent spectra and absorption spectra obtained in
oligo-DNAs T(20), T(6) and T(3) under the condition of |
5 a CuS04 concentration of 0.4 mM and an S.A.
concentration of 4 mM. The S.A. was added directly
before the measurements of the fluorescent spectrum and
the absorption spectra for the first time. After 8, 14,
24 and 35 minutes, the fluorescent spectra and the
I
10 absorption spectra were measured. The results are shown
in Figs. 7 and 8. In Fig. 7, the upper graphs each show I
the fluorescent spectrum with an ordinate axis of an
RFU value (absolute value), the middle graphs each show I
the fluorescent spectrum with an ordinate axis of an
15 RFU value (relative value) and the lower graphs each j
show the absorption spectrum. Fig. 8 shows a change
with elapsed time of the peak RFU value (A), and shows
a change with elapsed time at a wavelength of 34 6 nm
(B) .
20 [0055] As shown in the Figures, fluorescence is
almost disappeared in all oligo-DNAs of (T20), T(6) and
(T3) after 30 minutes. In particular, the fluorescence
is quickly disappeared in the oligo-DNAs having short
base lengths. After 35 minutes, the fluorescent spectra
25 were measured. Immediately thereafter, 1.8 ul of 44 mM
S.A. solution was again added to the sample for
39 SP306720WO00
j
measurement. The fluorescence could be again detected.
From this, the disappearance of the fluorescence could
be considered due to oxidation of Cu(I) ions to Cu(II)
ions. In each of the fluorescent spectra of the oligo-
5 DNAs T(6) and T(3), as the peak intensity was decreased,
a new peak was observed at a short wavelength side.
[0056] On the other hand, in each of the absorption
spectra of the respective oligo-DNAs, a decrease in the
peak intensity was observed with elapsed time. The
10 absorption spectra were more gradually decreased as
compared with the fluorescent spectra.
[0057] Fig. 9 (A) to (C) show two-dimensional
fluorescent spectra acquired in oligo-DNAs T(20), T(6)
and T(3) by the type F-4500 spectrofluorophotometer.
15 Fig. 10 shows excitation spectra (broken lines) and
fluorescent spectra (solid lines) obtained in the
respective oligo-DNAs. The spectrum was measured at a
space of 1 nm for a fluorescent wavelength, and at a
space of 2 nm for an excitation wavelength.
20 [0058] As shown in the Figures, it was confirmed
that the patterns of the fluorescent spectra were
changed depending on the base lengths of the oligo-DNAs.
It was also confirmed that the patterns of the
excitation spectra were changed depending on the base
25 lengths.
[0059] In order to further examine a relationship
40 SP306720WO00
between the base sequences and the spectra, the oligo- j
DNAs each having a three base length sequence by a
combination of adenine (A) and thymine (T) described in
SEQ ID NOS. 11 and 18 were measured for the
5 fluorescence. The results are shown in Figs. 11 and 12.
In Fig. 11, an ordinate axis (A) represents an RFU
value in each wavelength measured by the Nanodrop and
an ordinate axis (B) represents a value provided by
dividing the RFU value in each wavelength by a maximum
10 RFU value. Fig. 12 shows an average value and a
standard error by measuring the maximum value of the
RFU and the peak wavelength for three times.
[0060] As shown in the Figures, it was confirmed
that the fluorescence intensity and the peak wavelength
15 were changed depending on the base sequences of the
oligo-DNAs.
[0061] Fig. 13 shows the results of the measurement
obtained in the oligo-DNAs including sequence of SEQ ID
NOS. 19 and 20. It was confirmed that the oligo-DNAs
20 having the sequence described in SEQ ID NO. 20
containing uracil (U) emitted the fluorescence having
the spectrum shape and the peak position similar to
that of the oligo-DNAs having the sequence described in
SEQ ID NO. 19 containing thymine (T), although the
25 fluorescence intensity in the oligo-DNAs having the
sequence described in SEQ ID NO. 20 was faint as
41 SP306720WO00
compared with those having the sequence described in
SEQ ID NO. 19.
[0062]

5 This Example showed that the orange-colored
fluorescence having a wavelength of about 500 ran to 700
nm was observed by ultraviolet irradiation when DNAs
were mixed with a HEPPSO buffered solution containing
sodium chloride into which CuS04 and the S.A. were mixed.
10 It was confirmed that the fluorescence intensity
depended on the concentration of CUSO4, and the
fluorescence intensity and the spectrum were also
influenced by the base sequences of the nucleic acids.
[0063] The fluorescence was observed in the oligo-
15 DNAs containing at least thymine (T) , adenine (A) or
uracil (U) . In the experiment where the oligo-DNAs each
having a three base length containing thymine (T) and
adenine (A) were used, the fluorescence was observed in
any sequence. In addition, it is shown that the
20 fluorescence intensity and the spectra were influenced
not only by the amount of thymine (T) or adenine (A),
but also by the position (sequence order) on the oligo-
DNAs.
[0064] With time elapsed after the addition of the
25 S.A., the fluorescence intensity was decreased with
time, but was recovered by re-addition of the S.A. In
42 SP306720WO00
the meantime, Cu(I) ions are very unstable in the
presence of oxygen, and are changed into Cu(II) or
solid copper as soon as the reduction effect of the S.A.
is lost. From this, it is considered that the
5 fluorescence is derived from the composite of Cu(I)
ions and the nucleic acids. In order to detect the
fluorescence by the interaction between copper and the
nucleic acids, it may be desirable that a contact of
the reaction solution with oxygen in the air be
10 minimized.
[Example 2]
[0065] Example 2 illustrates that orange-colored
fluorescence similar to that observed in Example 1 was
emitted by ultraviolet irradiation under certain
15 conditions when a solution containing nucleic acids was
contacted with solid copper.
[0066]

As the copper that was contacted with the nucleic
20 acids, copper powder (Copper, Powder, -75um, 99.9% /
Cat.No.030-18352 / manufactured by Wako Pure Chemical
Industries, Ltd., Osaka, Japan) was used.
As the RNAs, Rat Brain Total RNAs (Cat.No.636622,
Takara Bio Inc., Otsu, Japan) were used by dissolving
25 it to DEPC treated water (Cat .No. 312-90201 / Wako Pure
Chemical Industries, Ltd., Japan).
43 SP306720WO00
PIPES, ACES, BES, TAPSO, HEPPSO, EPPS, TAPS, CAPS,
TES, Tricine and OPSO were purchased from DOJINDO
Laboratories (Kumamoto, Japan). Each of these was used
by adjusting the pH pursuant to the protocols provided
5 by the manufacturer. Other reagents were the same as in
Example 1.
[0067] The nucleic acids were contacted with copper
by mixing a variety of nucleic acids, salts and copper
powder into a total amount of 40 microliters solution,
10 and agitating it for 15 minutes. The amount of the
copper powder added was 375 mg per milliliter of the
solution, unless otherwise noted. The amount of the
salt, or sodium chloride (NaCl), was 500 mM, unless
otherwise noted.
15 [0068] After the sample was centrifuged to settle
the copper powder, a supernatant was measured for the
spectrum of the fluorescence and the intensity. The
measurement of the spectra of the fluorescence and the
intensity was performed in the similar steps as in
5 20 Example 1.
[0069]

The reaction solution to which 1.5 mg/ml of ssDNAs
were added was measured for the fluorescence three
25 times. The results are shown in Fig. 14 (abscissa axis:
wavelength, ordinate axis: RFU). As shown in the Figure,
i
44 SP306720WO00
when the sample containing the nucleic acids was
contacted with solid copper and then UV-excited, the
fluorescence having a peak around 600 nm could be
detected.
i
5 [0070] Next, a reaction solution was prepared by j
adding the copper powder in the amount of 375 mg, 250
mg, 125 mg, 62.5mg, 37.5 mg, 12.5 mg and 0 mg based on
1 mL of the reaction solution. To the reaction solution,
1.5 mg/ml of ssDNAs were added. The fluorescence was
10 measured for three times. The result was shown in Fig.
15. As shown in the Figure, the fluorescence intensity
depended on the amount of the copper powder. In the Cu
powder used in this Example, apparent fluorescence was
observed when the amount was 37.5 mg/ml or more. On the
15 other hand, no apparent fluorescence was observed when
the amount was 12.5 mg/ml or less.
[0071] Then, the type and the concentration of the
salt in the reaction solution were changed. To the
reaction solution, 1.5 mg/ml of ssDNAs were added. The
20 intensities of the fluorescence detected were compared.
The results are shown in Fig. 16. (A) shows the
fluorescence intensity detected in the reaction
solution to which 0.5, 0.25, 0.1, 0.05, 0.025 and 0 M
sodium chloride (NaCl) were added. (B) shows the
25 fluorescence intensity detected in the reaction
solution to which 0.45 M sodium chloride (NaCl), 0.45 M
45 SP306720WO00
potassium chloride (KC1), 0.45 M magnesium chloride
(MgC12) and 45% ethanol (EtOH) were added. The
fluorescence intensity was represented by the RFU at
604 nra, and measured for three times. The result was
5 shown as the average and the standard error. As shown
in the Figure, the fluorescence intensity depended on
the amount of sodium chloride. Also, the fluorescence
was detected under the coexistence of potassium
chloride and magnesium chloride as well as sodium
10 chloride.
[0072] Fig. 17 shows comparison results of the
fluorescence intensity detected when the concentration
of the nucleic acids added to the reaction solution was
changed. (A) shows the fluorescence intensity detected
15 in the reaction solution to which 5, 2.5, 1, 0.5, 0.25,
0.1, 0.05, and 0 mg/ml of ssDNAs were added. (B) shows
the fluorescence intensity detected in the reaction
solution to which 2.5, 0.25, and 0 mg/ml of RNAs were
added. The abscissa axis represents the concentration
20 of the nucleic acids, and the ordinate axis represents
the RFU at a fluorescent wavelength of 604 nm. The
measurement was performed for three times. The
concentration of sodium chloride (NaCl) was 0.25M, and
the amount of the copper power was 200 mg per 1 ml. The
25 condition was used in the following experiments, unless
otherwise noted. As shown in the Figure, the
i
46 SP306720WO00
fluorescence intensity depended on the concentration of
DNAs and the concentration of RNAs.
[0073] Next, the reaction solution to which 0.1 mM
oligo-DNAs having different sequences described in SEQ
5 ID NOS. 1, 2, 5, 6 and 9 was measured for the
fluorescence. The results are shown in Fig. 18. An
ordinate axis (A) represents an RFU value measured by
the Nanodrop, and an ordinate axis (B) represents a
relative RFU value when the peak height was set to 1.
10 As shown in the Figure, the fluorescence intensity and
the peak wavelength were influenced by the base
sequences. In particular, it could be confirmed that
when the percentage of thymine (T) was high, the
fluorescence intensity was high and the peak wavelength
15 became longer.
[0074] The reaction solution to which oligo-DNAs
having sequences described in SEQ ID NOS. 1, 2, 5 and 6
was also measured using the type F-4500
spectrofluorophotometer. Fig. 19 shows the results of
20 the fluorescent spectra (slit width of 2.5 ran) within
400 nm to 700 nm when the excitation light of 360 nm
(slit width of 10 nm) was irradiated. Again, it could
be confirmed that when the percentage of thymine (T)
was high, the fluorescence intensity was high and the
25 peak wavelength became longer in the sequence
| containing thymine (T) and adenine (A). Fig. 20 shows
1
I
|
I
?
•1
47 SP306720WO00
the results by scanning the excitation light at 330 nm
to 390 nm (slit width of 3 nm) and 400 nm to 700 nm
(slit width of 2.5 nm) to measure excitation -
fluorescent spectra. (A) represents three dimensionally,
5 and (B) represents a contour. An axis EX represents an
excitation wavelength (nm), an axis EM represents a
fluorescence wavelength (nm) and a height direction
represents the fluorescence intensity. Based on the
results, it could be read that the excitation, the
10 fluorescent spectra and the intensity were changed by
the different base sequences of the DNAs.
[0075] In order to further examine a relationship
between the base sequences and the spectrum, the oligo-
DNAs each having a combination sequence of cytosine (C)
15 having eight bases and thymine (T) having 12 bases
described in SEQ ID NOS. 21 and 26 were measured for
the fluorescence. The results are shown in Fig. 21. As
shown in the Figure, the fluorescence intensity
differed when the sequence was different even if the
20 base composition of the DNAs was the same.
[007 6] Next, the double-stranded DNAs including a
mismatch were measured for the pattern of the
fluorescent spectrum. As the double-stranded DNAs,
three types: a mixture ((e) + (f)) of oligo-DNAs each
25 having a sequence shown in SEQ ID NO. 1 and oligo-DNAs
each having a sequence shown in SEQ ID NO. 2, a mixture
48 SP306720WO00
((d) + (f)) of oligo-DNAs each having a sequence shown
in SEQ ID NO. 5 and oligo-DNAs each having a sequence
shown in SEQ ID NO. 2, and a mixture ((e) + (c) ) of
oligo-DNAs each having a sequence shown in SEQ ID NO. 1
5 and oligo-DNAs each having a sequence shown in SEQ ID
NO. 6 were used. Any of the oligo DNAs were mixed at a
final concentration of 0.5 mg/ml. The results are shown
in Fig. 22. An ordinate axis (A) represents an RFU '
value measured by the Nanodrop, and an ordinate axis
10 (B) represents a relative RFU value when the peak
height was set to 1. An abscissa axis represents a
wavelength (nm). As shown in the Figure, the
fluorescence intensity in the double-stranded DNAs was
lower than that in the single-stranded DNAs. However,
15 in the double-stranded DNAs having a mismatch of
thymine (T), the strong fluorescence was confirmed.
[0077] The intensities of the fluorescence detected
were compared, when the types of the buffer and the pH
in the reaction solution were changed. The results are
20 shown in Fig. 23. (A) shows relative values of peak RFU
values of a sample (+) containing ssDNAs and a sample
(-) containing no nucleic acids under each buffered
condition. (B) shows a relative value of a peak RFU
value of a sample containing oligo-DNAs having the
25 sequence shown in SEQ. ID NO. 1 under the same
condition. (C) shows a relative value of a peak RFU
0
49 SP306720WO00
value of a sample containing oligo-DNAs having the
sequence shown in SEQ. ID NO. 2 under the same
condition. The concentration of each buffer was 50 mM,
the final concentration of the ssDNAs was 0.5 mg/ml,
5 and the final concentration of the oligo-DNAs was 25 mM.
The relative value of the peak RFU value means that the
peak RFU value measured under no buffered condition is
set to 1. The fluorescence intensity depended on the
types of the buffer. The fluorescence was almost not
10 detected when no nucleic acids exist in the buffer.
[0078]

Based on the results in this Example, it revealed
that the fluorescence could be detected under adequate
15 conditions including the salt concentration, when the
nucleic acids were contacted with solid copper powder,
as is the case that the nucleic acids were contacted
with Cu(I) ions. It seemed that the fluorescence
observed in each case of copper ions and solid copper
20 was provided by the same mechanism, because their
properties such as wavelength properties and sequence
dependency are almost the same. Also, the fluorescence
was observed when the RNAs were used as the nucleic
acids. In addition, in the double-stranded DNAs, strong
25 fluorescence was observed when the mismatch exists
especially in thymine (T). This suggested that binding
50 SP306720WO00
with the complementary sequence might inhibit the
formation of the fluorescent substance by binding the
nucleic acids with copper. Also, it is considered that
the increase in the fluorescence intensity at the
5 mismatch site could be applied to a method of detecting
for mutation in the base sequences of the nucleic acids.
[007 9] In the experiments for comparing the
fluorescence under each buffered condition, the
fluorescence was observed in the buffer of PIPES, BES,
10 HEPPSO, EPPS, TAPS, CAPS, TES and POPSO. In particular,
strong fluorescence was detected in the buffer of PIPES,
HEPPSO, EPPS and POPSO. The fluorescence could be
observed within a pH range of 7.0 to 10.5. It was found
that a change in the fluorescence intensity depending
15 on the type of the buffer and the pH showed a different
pattern depending on the base sequences of the nucleic
acids. On the other hand, the buffer having a property
to chelate and stabilize Cu(II) ions has a tendency
that the fluorescence is not observed. Although no data
20 is provided in this Example, the fluorescence was
almost not observed when the reaction solution
containing, for example, a tris buffer, EDTA or the
like was used.
[Example 3]
25 [0080] In Example 3, it was confirmed that the
fluorescence could be detected after the nucleic acids
51 SP306720WO00
were brought into contact with copper sputtered on the
surface of the glass, and the properties of the
fluorescence were analyzed.
[0081]
5
As the DNAs, the ssDNAs described in Example 1
were used. As the RNAs, the RNAs described in Example 2
were used.
Copper was sputtered on the surface of the glass
10 using an apparatus, SH-350 manufactured by ULVAC, Inc.
(Kanagawa, Tokyo) on which a Cu target, 99.99% (Kojundo
Chemical Laboratory Co., Ltd, Saitama, Japan) was
mounted. In the sputtering, a thickness was set to 40
nm, and an adequate sputtering time was set based on a
15 deposition speed measured in advance. The glass for
sputtering silver was manufactured by Kyodo
International, Inc., Kanagawa, Japan.
[0082] On a slide glass on which copper or silver
was sputtered or an untreated slide glass, a sample
20 solution was placed, and a Gap cover glass, 24x25 NO.4
/ #CG00024 / Matsunami Glass Ind., Ltd., Osaka, Japan
was covered thereon. After it was allowed to be stood
for about 5 minutes, the fluorescence was observed. For
the observation, an inverted microscope Ti-U (Nikon Co.,
25 Tokyo, Japan) was used. For capturing the fluorescence,
a filter set UV-1A (Ex: 365/10, DM: 400, BA: 400/Nikon)
52 SP306720WO00
was used. For capturing and recording an image, a
digital CCD camera Retiga 2000R (Qlmaging, BC, Canada)
and a x20 objective lens were used.
[0083]
5
Fig. 24 shows images captured after the sample
containing 5 mg/ml of DNAs and 0.5 M of NaCl was
allowed to stand for 5 minutes on the copper sputtered
glass. Fig. 25 shows images captured after the sample
10 containing 5 mg/ml of RNAs and 0.5 M of NaCl was
allowed to stand for 5 minutes on the copper sputtered
glass.
[0084] As shown in Fig. 24(A), when the sample
containing DNAs was used, smooth fluorescence was
15 observed on the entire captured image. On the other
hand, as shown in Figs. 25 (A) and (B), when the sample
containing RNAs was used, the fluorescence having a
specific wave-like pattern within the captured image
was observed. A prospective cause of the pattern
20 specific to the RNAs was that the single-stranded RNAs
were hybridized each other to form the higher order
structure.
[0085] Next, the fluorescence intensity within the
captured image was converted into numerals. Each
25 captured image was divided into nine sections as shown
in Fig. 24 (B) . One of the nine sections (symbol C in
53 SP306720WO00
the Figure) was set to be a measuring range. An average
value of the fluorescence intensity within the
measuring range was calculated. For each sample, five
parts on the slide were captured to calculate the
5 average value from each image. The resultant five
average values were further averaged and calculated for
standard deviation.
[0086] Fig. 26 shows the fluorescence intensity
acquired when the sample containing the DNAs or the
10 RNAs was contacted with copper or silver sputtered on
the glass. In Fig. 26, "DNA/Cu", "RNA/Cu", and "{-)/Cu"
denote the sample containing 5 mg/ml DNAs, the sample
containing 5 mg/ml of RNAs, and the sample containing
no nucleic acids; the fluorescence intensity being
15 measured on the Cu sputtered glass. In
addition, "DNA/Ag", "RNA/Ag", and "(-)/Ag" denote the
sample containing 5 mg/ml of DNAs, the sample
containing 5 mg/ml of RNAs, and the sample containing
no nucleic acids; the fluorescence intensity being
20 measured on the Ag sputtered glass. Each sample
contained 0.5 M NaCl. Since the fluorescence intensity
in the "DNA/Cu" was significantly greater than those of
the other samples, its exposure time was 1 minute. In
all samples excluding the "DNA/Cu", the exposure time
25 was 5 seconds.
[0087] As shown in the Figure, in the Cu sputtered
54 SP306720WO00
glass, the "DNA/Cu" and the "RNA/Cu" had the
fluorescence intensity higher than the "(-)/Cu".
Especially in the DNA sample, the strong fluorescence
was detected. On the other hand, in the Ag sputtered
5 glass, the "DNA/Ag" and the "RNA/Ag" showed no increase
in the fluorescence intensity as compared with the "(-
)/Ag". As compared with the "(~)/Cu", the (-)/Ag"
showed the higher measured value. This may be caused by
a background derived from a reflected light, a
10 scattered light or autofluorescence on the Ag sputtered
surface.
[0088] Next, a change in the fluorescence intensity
with elapsed contact time of the nucleic acids with
copper was examined. A point of time when the sample
15 containing 5 mg/ml of ssDNAs and 0.5 M of NaCl was
placed between the Cu sputtered glass and the Gap cover
glass was designated as a starting point to measure the
fluorescence intensity per predetermined time. The
image was captured every 15 seconds, and a shutter for
20 excitation light was opened and closed per capturing
session. The lOx objective lens was used, and the
exposure time was 1 second. In every time, one image
captured was used to measure the fluorescence intensity.
The results are shown in Fig. 27.
25 [0089] As shown in the Figure, the fluorescence
intensity was gradually increased for several minutes
55 SP306720WO00
after the sample was introduced, and reached the
maximum value within about three minutes.
[0090] After a predetermined time was elapsed from
the contact of the nucleic acids with copper, a change
5 in the fluorescence intensity by temperature change was
examined. Immediately after the image has been captured,
it held at room temperature. After 50 seconds, a heat
block heated to 65°C was gently placed over the Cu
sputtered glass. After 100 seconds, the heat block was
10 removed. The image was captured every 5 seconds. After
150 seconds, the measurement was stopped for now and
the shutter for excitation light was closed. After 900
seconds, the measurement was again made. The results
are shown in Fig. 28.
15 [0091] As shown in the Figure, the fluorescence
intensity was gradually decreased for the first 50
seconds. This might be caused by fluorescence
photobleaching. During the next 50 seconds, the
fluorescence was disappeared at a speed apparently
20 different from the fluorescence photobleaching. After
the heat block was removed and it returned to room
temperature, the fluorescence was gradually recovered.
After 900 seconds, the fluorescence intensity was
returned to a level that color degraded fluorescence
25 intensity was subtracted from initial fluorescence
intensity. These results show that the fluorescence
i
56 SP306720WO00
emitted from the nucleic acids contacted with copper
was heat sensitive, and was reversibly disappeared as
the temperature increased.
[Example 4]
5 [0092] Example 4 illustrates that the cell nuclei
could be fluorescently observed by introducing the
sample containing cells onto the copper-sputtered glass.
[0093]

10 As PBS, Dulbecco's Phosphate Buffered Saline,
Ca/Mg free (Invitrogen Corporation, CA, USA) was used.
In an onion thin skin experiment, a commercially
available onion thin skin was carefully peeled by a
pair of tweezers, soaked into distilled water, rinsed
15 and used. The onion thin skin was placed on the Cu
sputtered glass, was soaked into the PBS, was covered
by a cover glass, and was then observed.
In the experiment of a human leukocyte sample,
IMMUNO-TROL Cells (Cat.No.6607077, Beckman Coulter,
20 Inc., Fullerton, CA, USA) were treated as follows:
Firstly, 500 microliters of the IMMUNO-TROL Cells were
separated, cleaned with PBS, and settled using a
centrifugal machine (1200rpm, 5min). Thereafter, a
supernatant was discarded to flake pellets, water
25 hemolysis treatments are repeated two times to provide
a sample. The sample was diluted with PBS, thereby
57 SP306720WO00
preparing a leukocyte sample. The water hemolysis
treatment was performed as follows: After the pellets ]
obtained as the result of the centrifugation were
sufficiently flaked, 9 ml of deionized water was added,
5 was mixed upside down for 30 seconds, 1 mL of lOx PBS
Buffer (Nippon Gene Co., Ltd., Tokyo, Japan) was added
and fully agitated. The cells were centrifuged (1200
rpm, 5 min) and were settled to remove a supernatant.
The leukocyte sample was placed on the Cu sputtered
10 glass, was covered by a cover glass, and was then
observed.
[0094] The copper sputtered glass, the cover glass,
the microscope etc. were the same as in Example 3. In
the sputtering, a thickness was set to 20, 40, or 100
15 nm. The thickness was set to 40 nm in the following
experiments, unless otherwise noted. When Cu was
sputtered only on a part of a slide glass surface, a
polyimide tape was adhered on the slide glass surface
excluding a 5mm square in a center part, thereby
20 performing the sputtering. Then, the polyimide tape was
removed. Thus, the Cu sputtered glass having a Cu layer
only formed on the 5 mm square in the center part was
produced.
[0095] The onion thin skin was fluorescently
25 observed using an excitation filter: 365/10 nm, a
dichroic mirror: 400 nm, and a fluorescent filter:
I
58 SP306720WO00
j
590LP. The leukocyte sample and Jurkat cells were
fluorescently observed using a filter set UV-1A (Ex:
365/10, DM: 400, BA: 400/Nikon).
[0096]
5
Fig. 29 shows images of the onion thin skin on the
copper sputtered glass fluorescently observed and
captured. (a) and (b) show observed images on the Cu
sputtered glass. (c) and (d) show observed images on a
10 slide glass without sputtering Cu thereon. (a) and (c)
are bright field observed images. (b) and (d) are
fluorescent images. (a) to (d) are images captured
using a lOx objective lens. (e) is an image captured
using a x40 objective lens.
15 [0097] As shown in the Figures, strong fluorescence
specific to the cell nuclei was observed on the cells
over the Cu sputtered glass. Although slight
fluorescence was observed on a part of cell walls and
the like, it is considered as autofluorescence of the
20 cell walls and the like, because it was observed on the
cells over the slide glass without sputtering Cu
thereon.
[0098] Next, animal cells were observed. Fig. 30
shows images acquired by fluorescently observing and
25 capturing the human leukocyte sample on the copper
sputtered glass. (a) is a bright field observed image.
I
59 SP306720WO00
(b) is a fluorescent image. The x40 objective lens was
used.
[0099] In the fluorescent image, segmented
neutrophils specific to the leukocyte were apparently
5 observed.
[0100] Fig. 31 shows images observed by using the Cu
sputtered glass where Cu was sputtered only on a part
of a slide glass surface. On the Cu sputtered glass,
human leukocyte cell strains, i.e., Jurkat cells, were
10 spread, were covered by the cover glass, and were then
observed using the x20 objective lens. The images were
captured at a boundary between a Cu deposited area and
a no Cu deposited area on the Cu sputtered glass. (a)
and (c) are bright field observed images; black areas
15 occupying more than half are areas where light is not
permeated, because the Cu layers are formed. (b) and
(d) are fluorescent images.
[0101] Strong fluorescence was observed only on the
cell nuclei of the cells in the Cu deposited area. Fig.
20 32 shows observation results of the Jurkat cells using
the Cu sputtered glass on which the Cu layer was formed
in a thickness of 20 nm (a) or 100 nra (b). The
fluorescence from the cell nuclei was observed at
either thickness.
25 [0102]

60 SP306720WO00
The results in this Example show that the
fluorescence can also be detected by bringing the cell
nuclei into contact with copper. It is clear that the
phenomenon occurs only on the glass substrate on which
5 copper is sputtered, and is the result of the action
between the cell nuclei and copper.
[0103] As a result of the fluorescence observation
of the onion thin skin cells and the leukocyte cells, a
difference between the shapes of cell nuclei in the
10 cells was apparently shown. From this, according to the j
method of detecting nucleic acids of the present
technology, different shapes of cell nuclei depending
on the types of the cells can be identified.
[0104] Although not shown in this Example, in the
15 experiment using the slide glass having copper
sputtered on a part thereof, after the fluorescence was
observed from only the cells on the Cu deposited area,
the slide glass was inclined to move the cells from the
Cu deposited area to the no Cu deposited area. After
20 moving, the fluorescence was continuously observed.
From this, even if the site where copper is contacted
with the cells is spaced from the site where the cells
are fluorescently observed, it is found that the
fluorescence can be detected by disposing a means for
25 moving the sample between the both sites.
[0105] After the fluorescence from the cell nuclei
61 SP306720WO00
of the cells between the Cu sputtered glass and the
cover glass was confirmed, the cover glass was removed
and the solution containing the cells were exposed to
air. Then, the fluorescence was quickly disappeared.
5 Also in the experiment using Cu(II) ions and S.A. in
Example 1, it was found that the fluorescence was
disappeared after the reaction solution was exposed to
air for a long time. The disappearance of the
fluorescence could be considered due to oxidation of
10 Cu(I) ions by the contact with air. Accordingly, the
fluorescence generation may be inhibited by bringing
the sample solution into contact with air (in
particular, exposing to oxygen contained in the air).
It is considered that the method of detecting nucleic
15 acids according to the present technology is preferably
performed by limiting the contact with air, e.g., in
the microchip.
[Example 5]
[0106] In Example 5, the cell nucleus observation
20 substrate according to the present invention was used,
and cells labeled with magnetic labeled antibodies were
selected and concentrated to observe the nuclear
fluorescence staining image.
[0107]
25
Substrate: Micro Slide Glass (Matsunami, Japan)
62 SP306720WO00
and Gap cover glass (Matsunami, Japan) were pressed. As
an absorption member, BEMCOT, M-3 (Asahi Kasei Corp.,
Japan) was inserted into the Gap cover glass at one end.
In the experiment using a copper sputtering, Cu was
5 sputtered on a surface of the Micro Slide Glass at 40
nm.
Magnetic labeled antibody: MACS CD45 MicroBeads
(hereinafter referred to as "MACS-CD45") was obtained
from Miltenyi Biotech GmbH (Germany). EasySep Human
10 CD45 Depletion Cocktail (hereinafter referred to as
"EasySep-CD45") was obtained from StemCell Technologies,
Inc. (Canada). EasySep-CD45 has stronger magnetism than
MACS-CD45.
Cell: Jurkat cells were used. A sample of the
15 cells was adjusted to be 1.75X107 cells/mL. To 200 ul
of the sample, 5 ul EasySep-CD45 or 10 ul of MACS-CD45
were added, which was incubated for 15 minutes. To the
EasySep-CD45 sample, 10 ul of an EasySep magnet reagent
was further added, which was incubated for 10 minutes.
20 In the experiment using a copper sputtering, the cells
were washed twice with PBS after incubation.
Magnetic adsorption of cells: The magnet used was
a neodymium magnet having a cylindrical shape with a
diameter of 5 mm and a height of 3 mm. The magnet was
25 disposed in contact with an upper surface of the Gap
cover glass. The cells labeled with the magnetic
j
63 SP306720WO00
substance flowed into a gap having a depth of 20
micrometers formed between a lower surface of the Gap
cover glass and the slide glass, whereby the cells
labeled with the magnetic substance were accumulated
5 around the magnet disposed.
[0108] Fig. 33 shows photographs of EasySep-CD45
labeled cells accumulated on the observation area of
the substrate, and Fig. 34 shows photographs of MACSCD45
labeled cells. In the Figures, at left hand, the
10 introduction part into which the sample liquid is
introduced is positioned. At right hand, the absorption
member for absorbing the sample is positioned. At
center, the observation area to which the magnet is
disposed is positioned. The photographs were taken
15 after the magnet was removed. It was confirmed that the
EasySep-CD45 labeled cells and the MACS-CD45 labeled
cells were accumulated on the observation area
corresponding to the position at which the magnet was
disposed. The lower photographs were magnified
| 20 photographs of a part (an introduction part side) of
the observation area.
[0109] Fig. 35 shows a permeated image (A) captured
of the MACS-CD45 labeled cells accumulated on the
observation area of the substrate on which copper was
25 sputtered, and a fluorescence image (B) acquired by
irradiating the ultraviolet rays. In the fluorescence
64 SP306720WO00
image (B), the cell nuclei accumulated show
fluorescence, and nucleic shapes are obviously observed.
[Industrial Applicability]
[0110] By the cell nucleus observation substrate 5 according to the present invention, nuclei of cells,
microorganisms and the like can be stained by a simple
operation and observed their forms. Accordingly, the
cell nucleus observation substrate according to the
present invention can be effectively used for
10 determination of types or properties of cells,
microorganisms, and the like based on forms of cell
nuclei in a variety of fields including a medical field,
a drug discovery field, a food field, an agricultural
field and the like.
Description of Symbols I
[0111]
A cell nucleus observation apparatus
1 introduction part
20 2 observation area
3 copper film
4 absorption member
5 magnet
6 outlet
25 ai, a2 substrate layer
a3 spacer
SEQUENCE LISTING

65 SP306720WO00
Claims
[1] A cell nucleus observation substrate, comprising:
an introduction part into which a sample liquid
containing a cell is introduced; and
5 an observation area within which the cell in the
sample liquid introduced from the introduction part is
held,
copper being disposed on a flow path of the sample
liquid in the introduction part and the observation
10 area so that the copper being capable of contacting
with the sample liquid.
[2] The cell nucleus observation substrate according to
claim 1, wherein
the copper is preferably formed and disposed on
15 the flow path.
[3] The cell nucleus observation substrate according to
claim 2, comprising:
a magnet that is disposed attachably and
detachably and forms a magnetic field within the
20 observation area, wherein
the cell in the sample liquid flowing through the
flow path is held within the observation area based on
a magnetic force.
[4] The cell nucleus observation substrate according to
25 claim 3, wherein:
the observation area is disposed in a space
66 SP306720WO00
between two opposing substrates at a predetermined
distance,
comprising:
an introduction part where the sample liquid is
5 introduced into the space; and
an absorption member that absorbs the sample
liquid introduced from the introduction part to the
space, wherein
the magnet is attached at a position between the
10 introduction part and the absorption member.
[5] A cell nucleus observation apparatus, comprising:
an introduction part into which a sample liquid
containing a cell is introduced;
an observation area within which the cell in the
15 sample liquid introduced from the introduction part is
held;
a substrate where copper is disposed on a flow
path of the sample liquid in the introduction part and
the observation area so that the copper being capable
20 of contacting with the sample liquid; and
an optical detecting means for irradiating a light
to the observation area and detecting fluorescence
generated.
[6] The cell nucleus observation substrate according
25 to claim 5, wherein:
a wavelength of the light irradiated by the
,' 67 SP306720WO00
optical detecting means is 300 to 420 nm.