U.S. patent application number 11/064828 was filed with the patent office on 2005-06-30 for electrode for dielectrophoretic apparatus, dielectrophoretic apparatus, method for manufacturing the same, and method for separating substances using the electrode or dielectrophoretic apparatus.
This patent application is currently assigned to WAKO PURE CHEMICAL INDUSTRIES, LTD.. Invention is credited to Kawabata, Tomohisa, Washizu, Masao.
Application Number | 20050139473 11/064828 |
Document ID | / |
Family ID | 26590060 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050139473 |
Kind Code |
A1 |
Washizu, Masao ; et
al. |
June 30, 2005 |
Electrode for dielectrophoretic apparatus, dielectrophoretic
apparatus, method for manufacturing the same, and method for
separating substances using the electrode or dielectrophoretic
apparatus
Abstract
To provide an electrode for a dielectrophoretic apparatus in
which a background detected by reflecting an excited light on an
electrode present under the substance (molecule) is reduced and an
S/N ratio is enhanced. Also, there is provided an dielectrophoretic
apparatus, in an apparatus in which a liquid containing substances
to be separated is present in a non-uniform electric field formed
by a dielectrophoretic electrode, and separation is carried out by
a dielectrophoretic force exerting on the substances, wherein the
collecting ability of substances is enhanced. The present invention
is characterized in that a vacant space is provided in an electrode
whereby substances subjected to influence by a negative
dielectrophoretic force can be concentrated in said vacant space of
an electrode, or above or below portion of the space.
Inventors: |
Washizu, Masao; (Sakyo-ku,
JP) ; Kawabata, Tomohisa; (Amagasaki-Shi,
JP) |
Correspondence
Address: |
ARMSTRONG, KRATZ, QUINTOS, HANSON & BROOKS, LLP
1725 K STREET, NW
SUITE 1000
WASHINGTON
DC
20006
US
|
Assignee: |
WAKO PURE CHEMICAL INDUSTRIES,
LTD.
Osaka
JP
|
Family ID: |
26590060 |
Appl. No.: |
11/064828 |
Filed: |
February 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11064828 |
Feb 25, 2005 |
|
|
|
09833566 |
Apr 13, 2001 |
|
|
|
6875329 |
|
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Current U.S.
Class: |
204/643 ;
204/547 |
Current CPC
Class: |
B03C 5/026 20130101 |
Class at
Publication: |
204/643 ;
204/547 |
International
Class: |
B01D 057/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2000 |
JP |
112337/2000 |
Dec 8, 2000 |
JP |
374210/2000 |
Claims
What is claimed is:
1. An electrode for a dielectrophoretic apparatus characterized in
that a vacant space is formed in the electrode in such a way as
concentrating substances subjected to influence by a negative
dielectrophoretic force in said vacant space of the electrode or
above or below position of the space.
2. The electrode according to claim 1 wherein said vacant space is
a hollow space.
3. The electrode according to claim 1 wherein all the circumference
of said vacant space is surrounded by the electrode.
4. The electrode according to claim 1 wherein an area in which
substances subjected to influence by a negative dielectrophoretic
force are concentrated is an area in which density of an electric
flux line is low.
5. The electrode according to claim 1 wherein said electrode is
formed in such a way as concentrating substances subjected to
influence by a negative dielectrophoretic force in said electrode
when a liquid containing said substances subjected to influence by
a negative dielectrophoretic force is positioned at said electrode
or above the vacant space or in the vicinity thereof, or is caused
to flow above or below thereof.
6. The electrode according to claim 1 wherein said electrode is in
the form of circular, oval or polygonal, and a circular, oval or
polygonal vacant space is formed in the central part thereof.
7. The electrode according to claim 1 wherein said electrode is
provided on a substrate.
8. The electrode according to claim 7wherein said substrate
provided with said electrode is made of a material which does not
substantially reflect excitation light or permeates light to such
an extent as capable of measuring absorbance.
9. The electrode according to claim 8wherein said substrate
provided with said electrode is made of a transparent material.
10. The electrode according to claim 1 wherein the substances
subjected to influence by the negative dielectrophoretic force
generated by application of voltage to said electrode are granular
substances.
11. An electrode construction for an dielectrophoretic apparatus
comprising an electrode and a lid provided thereabove so as to form
a gap between the lid and said electrode surface, wherein a vacant
space is formed in the electrode in such a way as concentrating
substances subjected to influence by a negative dielectrophoretic
force generated by application of voltage to said electrode in a
vacant space of said electrode or above or below position of the
space.
12. A method for manufacturing an electrode according to claim 1
characterized in that said vacant space is formed by physical or
chemical means.
13. The method for manufacturing an electrode according to claim 1
wherein said electrode and said vacant space are prepared by the
fine processing technique.
14. A dielectrophoretic apparatus comprising the electrode for a
dielectrophoretic apparatus of claim 1.
15. A dielectrophoretic apparatus characterized in that in a
dielectrophoretic apparatus provided with an electrode on a
substrate, a construction for realizing an increase of non-uniform
electric field region is formed among electrodes.
16. A dielectrophoretic apparatus characterized in that in a
dielectrophoretic apparatus provided with an electrode on a
substrate, the places among said electrodes are made in lower level
than the electrode level.
17. The dielectrophoretic apparatus according to claim 16 wherein
said electrode is held by a convex construction on said substrate
to make the places among said electrodes in lower level than said
electrode level.
18. A method for manufacturing a dielectrophoretic apparatus
characterized in that a substrate is excavated by physical or
chemical means to make the places among said electrodes in lower
level than said electrode level.
19. The method for manufacturing a dielectrophoretic apparatus
according to claim 18 wherein said chemical means is an etching
using an etching liquid for the substrate of said dielectrophoretic
apparatus.
20. In a separation method in which a liquid containing substances
to be separated is present within non-uniform electric field
generated by a dielectrophoretic electrode, and separation is
carried out by utilizing difference in the dielectrophoretic forces
exerting on said substances, the improvement is that an increase of
non-uniform electric field is realized by making the places among
the electrodes in lower level than the electrode level, so as to
enhance the collecting ability of substances.
21. In a separation method in which a liquid containing substances
to be separated is caused to flow into non-uniform electric field
generated by the dielectrophoretic electrode, and separation is
carried out by an interaction of the dielectrophoretic force
exerting on said substance and fluid drag, the improvement is that
the increase of non-uniform electric field region and the reduction
in fluid drag are realized by making the places among the
electrodes in lower level than the electrode level, so as to
enhance the collecting ability of substances.
22. A dielectrophoretic apparatus comprising the electrode
construction for a dielectrophoretic apparatus of claim 11.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a division of Ser. No. 09/833,566, filed
Apr. 13, 2001, which is based on Japanese Application No.
2000-112337 filed Apr. 13, 2000 and No. 2000-374210 filed Dec. 8,
2000.
BACKGROUND OF THE INVENTION
[0002] This invention relates to an electrode for a
dielectophoretic apparatus, in which a background can be reduced to
enhance an S/N (Signal/Noise) ratio in detecting a substance to be
measured (molecules to be measured) by a fluorescent strength or
the like, a method for manufacturing the same, an electrode
constitution provided with the electrode, and a method for
separating substances using the electrode.
[0003] This invention further relates to an dielectrophoretic
apparatus having an enhanced collecting ability, a method for
manufacturing the same, and a method for separating substances
using the apparatus.
[0004] Processing technology of materials at scales of nanometer to
micrometer by means of micromachining technology such as
photolithography has recently been established by development of
semiconductor technologies and it has still continued its progress
at present.
[0005] In the fields of chemistry and biochemistry, new technology
called a Micro Total Analysis System (.mu.-TAS), Laboratory on a
chip is growing, in which such micromachining technology is
employed to carry out a whole series of chemical/biochemical
analytical steps of extraction of component (s) to be analyzed from
biological samples (extraction step), analysis of the component(s)
with chemical/biochemical reaction(s) (analysis step), and
subsequent separation (separation step) and detection (detection
step) using a highly small analytical device integrated on a chip
having each side of a few centimeters to a few ten centimeters in
length.
[0006] Procedures of the .mu.-TAS are expected to make a large
contribution to saving the analyzing time, reducing the amounts of
samples to be used and reagents for chemical/biochemical reactions,
and reducing the size of analytical instruments and the space for
analysis in the course of all the chemical/biochemical analytical
steps.
[0007] For the separation step in .mu.-TAS, in particular, there
have been developed capillary electrophoretic methods in which a
capillary (fine tube) with an inner diameter of less than 1 mm
which is made of Teflon, silica, or the like as material is used as
the separating column to achieve separation with charge differences
of substances under a high electric field, and capillary column
chromatographic methods in which a similar capillary is used to
achieve separation utilizing the difference of the interaction
between carrier in the column medium and substances.
[0008] However, capillary electrophoretic methods need a high
voltage for separation and have a problem of a low sensitivity of
detection due to a limited capillary volume in the detection area
and also these is found such a problem that they are not suitable
for separation of high molecular weight substances, though suitable
for separation of low molecular weight substances, since the length
of capillary for separation is limited on the capillary column on a
chip and thus a capillary can not be made into a length enough for
separating high molecular weight substances. In addition, in
capillary column chromatographic methods there is a limit in making
the throughput of separation processing higher and also there is
such a problem that reducing the processing time is difficult.
[0009] Thus, attention has recently been paid to a method for
solving the problems as described above, which comprises utilizing
such a phenomenon so-called dielectrophoretic force that a positive
and negative polarization occurs in substances placed under a
non-uniform electric field, thereby providing a driving force of
moving the substances [H. A. Pohl, "Dielectrophoresis", Cambridge
Univ. Press (1978); T. B. Jones, "Electromechanics of Particles",
Cambridge Univ. Press (1995), and the like].
[0010] These separation methods are presently believed to be the
suitable separation method in .mu.-TAS from the following points:
(1) a rapid separation can be expected at a low applied voltage
without requiring a high voltage as in capillary electrophoresis,
since an electric field and its gradient can be increased to an
extreme extent if micromachined electrodes are employed, because
the degree of dielectrophoretic forces depends on the size and
dielectric properties of substances (particles) and is proportional
to the electric field gradient; (2) an increase in temperature due
to applying the electric field can be minimized, since a strong
electric field area is localized at a significantly small region,
and a high electric field can be formed; (3) as the
dielectrophoretic force is a force proportional to the electric
field gradient, the force is understood as independent on the
polarity of the applied voltage, and thus works under an AC
electric field in a similar way to a D.C. electric field, and
therefore if a high frequency A. C is employed, an electrode
reaction (electrolytic reaction) in an aqueous solution can be
suppressed, so that the electrodes themselves can be integrated in
the channel (sample flow path); (4) improvement in a detection
sensitivity can be expected, since there is no restriction to a
chamber volume of the detection component unlike the capillary
electrophoresis, and the like.
[0011] The dielectrophoresis termed herein is a phenomenon in which
neutral particles move within non-uniform electric field, and the
force exerting on molecules is called a dielectrophoretic force.
The dielectrophoretic force is divided into two forces, i.e., a
positive dielectrophoretic force in which substances move toward a
high electric field, and a negative dielectrophoretic force in
which substances move toward a low electric field.
[0012] (General Equation of Dielectrophoretic Forces)
[0013] The equivalent dipole moment method is a procedure of
analyzing dielectrophoretic forces by substituting induced charges
for an equivalent electric dipole. According to this method, the
dielectrophoretic force F.sub.d acted upon a spherical particle
with a radius of a which is placed in an electric field E is given
by:
F.sub.d=2.pi.a.sup.3.epsilon..sub.mRe[K*(.omega.)].gradient.(E.sup.2)
(1)
[0014] wherein K*(107 ) means by using an angular frequency of the
applied voltage .omega. and the imaginary unit j as follows:
K*(.omega.)=.epsilon..sub.p*-.epsilon..sub.m*/.epsilon..sub.p*+2.epsilon..-
sub.m* (2)
.epsilon..sub.p*=.epsilon..sub.p-j.sigma..sub.p/.omega.,
.epsilon..sub.m*=.epsilon..sub.m-j.sigma..sub.m/.omega. (3)
[0015] wherein .epsilon..sub.p, .epsilon..sub.m, .sigma..sub.p,
.sigma..sub.m are permittivity and conductivity of the particle and
the solution, and complex quantities are designated by *.
[0016] Equation (1) indicates that in a case of
Re[K*(.omega.)]>0, the force works in such a way as attracting
the particle toward a strong electric field side (positive
dielectrophoretic, positive DEP), and in a case of
Re[K*(.omega.)]<0, the force works in such a way as pushing the
particle toward a weak electric field side (negative
dielectrophoretic, negative DEP).
[0017] As will be apparent from the above-described Equations,
whether the positive electrophoresis occurs in a certain substance
or the negative electrophoresis occurs therein is decided by the
interaction of three parameters, i.e., 1) frequency of an electric
field applied, 2) conductivity and permittivity (dielectric
constant) of medium, and 3) conductivity and permittivity
(dielectric constant) of substance.
[0018] When these parameters are changed, even the same substance
shows a positive dielectrophoresis or a negative dielectrophoresis.
The negative dielectrophoresis is a phenomenon in which the
substance moves toward a low electric field which is weak in
density of electric flux line while the positive dielectrophoresis
moves toward a high electric field which is high in density of
electric flux line. FIG. 1 is a view for explaining the negative
dielectrophoresis. The negative dielectrophoretic force is a force
for carrying substances to such a field as to be lowered where the
density of electric flux line received by the substance.
[0019] Sometimes, the substances are measured by concentrating them
in an area where an electric field on an electrode is weak by using
the negative dielectrophoresis as described and thereafter
measuring them by fluorescent strength or the like. The detection
of the fluorescent strength is carried out by irradiating an
excitation light on the substance to be measured to observe
fluorescent light from the upper surface of the electrode.
[0020] At that time, where a conventional electrode is used, there
poses a problem that the excitation light is reflected even on the
electrode which is present under the substance to be measured, and
thus reflected light is detected as a great background. This leads
to a problem of reducing the measurement sensitivity. Besides,
where a conventional electrode is used, since light does not
permeate through the electrode, the substances concentrated
(gathered ) on the electrode cannot be detected by absorbance.
[0021] Further, the dielectrophoresis is contemplated to be a
separation method suitable for .mu.-TAS. However, In consideration
of a case of application of the dielectrophoresis to .mu.-TAS, it
is extremely important to enhance the collecting ability. In this
respect, the conventional dielectrophoretic apparatus should not
yet be satisfied.
[0022] That is, if the collecting ability of substances is
enhanced, separation becomes enabled in the electrode region, and
the substances are held efficiently, whereby separation with high
S/N (Signal/Noise) ratio is realized. Further, for example,
particularly, in the Field-Flow fractionation for carrying out
separation by the interaction of the dielectrophoretic force and
the fluid drag exerting on the substances, separation in a short
electrode region can be made even at the same flow velocity.
SUMMARY OF THE INVENTION
[0023] [Invention 1]
[0024] It is an object of the present invention to provide an
electrode for a dielectrophoretic apparatus which reduces a
background in which an excitation light is reflected on an
electrode which is present under a substance (a molecule) and
detected to enhance an S/N ratio.
[0025] It is a further object of the present invention to provide
an electrode for a dielectrophoretic apparatus, which can be
detected even by absorbance.
[0026] It is another object of the present invention to provide a
method for separating substances and a detection method using the
above electrode.
[0027] For achieving the aforementioned objects, the present
inventors have studied earnestly, as a result of which the
inventors have thought out that an electrode in an area where
substances to be measured are concentrated (gathered) is removed to
thereby enable reduction in background caused by reflection of an
excitation light from the electrode.
[0028] In the past, there are many patents and articles in
connection with apparatus and method in a dielectrophoretic
chromatography apparatus (Field-Flow fractionation), but a
dielectrophoretic apparatus and method which reduces a background
by removing an electrode including an area where substances to be
measured are concentrated to enhance an S/N ratio are not known at
all, and such an idea is not known at all.
[0029] The present invention is characterized in that by forming a
vacant space in an electrode, substances subjected to influence by
a negative dielectrophoretic force generated by application of
voltage to the electrode are concentrated in the vacant space of
the electrode, or above or below position of the space.
[0030] The vacant space is formed from a hollow space or formed of
a material which does not substantially reflect excitation light or
permeates light to such an extent as capable of measuring the
absorbance. However, the vacant space is preferably a hollow
space.
[0031] The space where substances subjected to influence by the
negative dielectrophoretic force are concentrated is a space in
which the density of electric flux line is low for the
substances.
[0032] Further, through all the substances subjected to influence
by the negative dielectrophoretic force are preferably concentrated
in the vacant space, concentrated substances in the vacant space
may be a part of all the substances.
[0033] The electrode constitution of the present invention is
characterized by comprising an electrode, and a lid provided
thereabove so as to form a gap between the lid and said electrode
surface, the electrode being formed as in the electrode of the
present invention provided with the vacant space.
[0034] The electrode constitution of the present invention includes
an electrode of the present invention, a substrate (an electrode
base plate) and a lid. In the dielectrophoretic apparatus, a device
for applying a voltage to an electrode and a detection section are
added to the electrode or the electrode constitution.
[0035] A method for manufacturing an electrode according to the
present invention characterized in that said vacant space is formed
by physical or chemical means.
[0036] The separation method and detection method according to the
present invention are characterized in that using the electrode of
the present invention provided with the vacant space, a liquid
including substances subjected to influence by the negative
dielectrophoretic force generated by application of voltage to the
electrode is positioned in the electrode or the vacant space or in
the vicinity thereof, or causes to flow thereabove or therebelow,
whereby substances subjected to influence by the negative
dielectrophoretic force are concentrated(gathered) in the vacant
space, or above or below position of the space.
[0037] The separation method of the present invention can be used
for liquids in which two kinds or more of substances are dissolved
or suspended, but preferably, the substances subjected to influence
by the negative dielectrophoresis force concentrated in the vacant
space or in a vertical direction thereof are granular substances.
Because, in the granular substances, an area in which the density
of electric flux line is low and the granular substances are
concentrated tends to be the vacant space or in a vertical
direction thereof.
[0038] The vacant space of the present invention, should be formed
in such a way that an area in which the density of a electric flux
line is low and the granular substances are concentrated may be
formed in the vacant space or in a vertical direction thereof by
changing the size of the substances subjected to influence by the
negative dielectrophoresis force, and the width and depth of an
electrode used (the height from the electrode surface to the lid
part and or the height from the vessel bottom to the electrode
surface) and frequently applied.
[0039] However, particularly, where the substances to be measured
are dissolved, for example, in liquid such as water, preferably,
the substances subjected to influence by the negative
dielectrophoresis force are bound to the substances to be measured
in a sample through "substances binding to the substances to be
measured" to form a complex, and a reaction substance including the
complex is applied to the dielectrophoresis.
[0040] It is noted that the substances to be measured used in the
present invention means substances (molecules) to be concentrated
in the area in which the density of electric flux line is low, and
need not always be an object for measurement.
[0041] [Invention 2]
[0042] It is a further object of the present invention to provide,
in an apparatus for enhancing the collecting ability of substances
in which a liquid containing substances to be separated is present
within a non-uniform electric field formed by a dielectrophoretic
electrode to separate the substances by the dielectrophoretic force
exerting on the substrate.
[0043] For achieving the aforementioned objects, the present
inventors have studied earnestly, as a result of which the
inventors have thought out that a base plate (substrate) of among
electrodes are excavated to form a part lower than the electrode
level whereby the non-uniform electric field region is increased
and the drag of fluid is reduced to enhance the collecting
ability.
[0044] In the past, there are many patents and articles in
connection with separation apparatus and method making use of a
dielectrophoretic force, particularly, apparatus and method in
Field-Flow fractionation, but an apparatus and method which
enhances the collecting ability by forming "a lower level place
than an electrode level" are not known at all, and such an idea is
not known at all.
[0045] Preferably, the present invention provides a
dielectrophoretic apparatus having an electrode provided on a
substrate, wherein means for realizing an increase of an
non-uniform electric field region is formed among the
electrodes.
[0046] The means for realizing an increase of a non-uniform
electric field region is characterized in that a lower level places
than the electrode level is formed among the electrodes. The "lower
level place than the electrode level" is formed whereby electric
fields are formed not only above between the electrodes but below
thus increasing a non-uniform electric field region, and further,
where for example, Field Flow fractionation is used, since the flow
velocity of fluid in that places drops, the fluid drag is reduced
to enhance the collecting ability of substances.
[0047] For forming "lower level places than electrodes level", a
base plate (substrate) may be excavated between electrodes by
physical and/or chemical means to form the lower level place than
the electrode level among the electrodes. The physical means termed
herein is, for example, a method for excavation using a suitable
knife or the like, for example, an LIGA (Lithographile
Galvanoformung Abformung) method using synchrotron radiant light.
Further, the chemical means is etching for excavating a base plate
using an etching liquid for a base plate. Further, for example a
base plate can be excavated by etching using plasma of a reaction
gas [Reactive ion etching (RIE)] formed by a high frequency power
supply, in which a physical excavation and chemical excavation are
conducted at the same time. It is noted that the means as described
above may be suitably combined to carry out excavation of a base
plate.
[0048] Further, a separation method according to the present
invention is a separation method for substances in which a liquid
containing substances to be separated is present within a
non-uniform electric field formed by the dielectrophoretic
electrode, and separation is carried out due to a difference in a
dielectrophoretic force exerting on the substances characterized in
that an increase of a non-uniform electric field region is realized
by lower level places than electrode level formed between (or
among) electrodes, to thereby enhance the collecting ability.
[0049] Dielectrophoresis (DEP) termed herein is a phenomenon in
which a neutral particle moves within a non-uniform electric field
by interaction of conductivity and dielectric constant of
substances, conductivity and dielectric constant of media, and
frequency applied, and a force acting on the particle is called a
dielectropherotic force. The dielectrophoretic force is divided
into two kinds, i.e., a positive dielectrophoretic force in which
substances move toward a high electric field, and a negative
dielectrophoretic force in which substances move toward a low
electric field.
[0050] In the following, a case where a positive dielectrophoretic
force exerts on a molecule will be described.
[0051] Namely, as shown in FIG. 2, a neutral molecule placed in an
electric field has a positively induced polarization charge +q
downstream in the electric field and a negatively induced
polarization charge -q upstream in the electric field,
respectively, thus +q receives a force of +qE from the electric
field E and this portion is pulled upstream in the electric field.
If the molecule is neutral, +q and -q have an equal absolute value,
and if the electric field is uniform regardless of the positions,
both received forces are balanced, therefore the molecule does not
move. However, in the case where the electric field is non-uniform,
an attractive force toward a strong electric field becomes larger,
thus the molecule is driven toward the strong side of the electric
field.
[0052] As described above, the molecule in a solution variously
moves within an electric field according to the dielectrophoretic
force generated in the molecule. However, for example, in the
Field-Flow fractionation, the movement of molecules is governed by
three factors: the dielectrophoretic force F.sub.d, the force
F.sub.v generated by the drag due to the flow in the flow path, and
the force F.sub.th due to the thermal movement. {circle over (1)}
in the case of F.sub.d>>F.sub.v+F.sub.th, molecules are
captured (trapped) on the electrode, {circle over (2)} in the case
of F.sub.d<<F.sub.v+F.sub.- th, molecules are eluted out with
flow in the flow path, regardless of the electric field. {circle
over (3)} in the case of F.sub.d.apprxeq.F.sub.v+- F.sub.th,
molecules are carried downwards with repeating adsorption and
desorption on the electrode, so that the molecules arrive at the
outlet with delay, relative to the set flow in the flow path.
[0053] In the present invention, since a portion between electrodes
is excavated deeply whereby a non-uniform electric field is formed
below between the electrodes, the non-uniform electric field region
is increased and the flow of fluid in that portion becomes slow to
reduce the drag force Fv of fluid, whereby Fd becomes further great
under the condition {circle over (1)} as described above and Fv
becomes further small thus enhancing the collecting rate. Further,
the particles trapped in the electric field formed below between
electrodes are hard to flow out since the particles are positioned
at "lower level places than electrode level".
[0054] The above and other objects and advantages of the invention
will become more apparent from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1 is an explanatory view of the negative
dielectrophoresis.
[0056] FIG. 2 is a view showing the principle of the positive
dielectrophoresis.
[0057] FIG. 3 is a plan view showing an embodiment of an electrode
of the present invention.
[0058] FIG. 4 is a plan view showing a further embodiment of an
electrode of the present invention.
[0059] FIG. 5 is a plan view showing another embodiment of an
electrode of the present invention.
[0060] FIGS. 6A and 6B are plan views showing examples of a
conventional electrode.
[0061] FIGS. 7A and 7B are plan views showing further examples of a
conventional electrode.
[0062] FIGS. 8A and 8B are plan views showing other examples of a
conventional electrode.
[0063] FIGS. 9A and 9B are plan views showing still other examples
of a conventional electrode.
[0064] FIGS. 10A and 10B are plan views showing other examples of a
conventional electrode.
[0065] FIGS. 11A through 11G are plan views showing still other
examples of a conventional electrode.
[0066] FIG. 12 is an explanatory view in the case where fluorescent
measurement is made according to the method of the present
invention, (A) showing the case where a fluorescent measuring unit
is provided above, (B) showing the case where a fluorescent
measuring unit is provided below.
[0067] FIG. 13 is a plan view showing an electrode of the present
invention prepared in Example 1.
[0068] FIG. 14 are respectively, a plan view (A) and a sectional
view (B) showing a further embodiment of the present invention.
[0069] FIG. 15 is a sectional view showing an example of "lower
level places than electrode level" of the present invention formed
by isotropic etching (A), anisotropic etching (B), and RIE or LIGA
(C),
[0070] FIGS. 16A through 16E are plan views showing electrodes used
in the present invention.
[0071] FIG. 17 is a sectional view of a dielectrophoretic
chromatography apparatus.
[0072] FIG. 18 is a sectional view showing an example of forming
"lower level place than electrode level" on a base plate
(substrate) according to the method of the present invention.
[0073] FIG. 19 is a graph showing a relationship between etching
time and the depth of a groove measured in Example 3.
[0074] FIG. 20 is a graph which measured the collecting rate with
respect to bovine-serum albumin (BSA) protein, using the
dielectrophoretic chromatography apparatus according to the present
invention and the conventional dielectrophoretic chromatography
apparatus.
[0075] FIG. 21 is a graph which measured the collecting rate with
respect to 500 bp DNA, using the dielectrophoretic chromatography
apparatus according to the present invention and the conventional
dielectrophoretic chromatography apparatus.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0076] The preferred embodiments of the present invention will be
described hereinafter.
[0077] First, the invention 1 will be described in detail
hereinafter.
[0078] FIG. 3 is a plan view showing an embodiment of an electrode
for the dielectrophoretic apparatus of the present invention,
showing an example in which a hollow space (a vacant space) 12 is
formed in a part 13 on which are concentrated substances
(substances to be measured) subjected to influence by the negative
dielectrophoretic force generated by an electrode 11 having many
hexagonal portions associated.
[0079] The hollow space 12 is formed so as to form an area which is
low in density of electric flux line in which the substances to be
measured may be concentrated in the hollow space 12 or in a
vertical direction thereof. The area which is low in density of
electric flux line is an area which is lower in density of electric
flux line than that of an electrode in the circumference, and in
general, an area which is lowest in density of electric flux line.
The size of the hollow space 12 is different depending on the kind
and size of substances to be measured, the distance between an
electrode base plate and a cover glass (depth) or the like, but is
generally formed to be larger than a space 13 on which are
concentrated the substances to be measured when the hollow space is
not formed. The hollow space 12 may be communicated as shown in
FIG. 3 or may be independent every hexagonal portion as shown in
FIG. 4.
[0080] In the hollow space 12, all the circumference may be
surrounded by the electrode or a break 14 may be present in a part
as shown in FIG. 3, but preferably, all the circumference may be
surrounded by the electrode.
[0081] When all the circumference of the vacant space is surrounded
by the electrode, electric flux lines are generated from the
circumference of the vacant space, and therefore, the vacant space
is to be surrounded by a high electric field region so that the
substances tend to be concentrated on a specific portion and may be
collected easily.
[0082] On the other hand, where a space of the vacant space is not
surrounded by the electrode, no line of electric force is generated
from that portion, and therefore, a portion which is not a high
electric field region is generated, and the substances may be
easily moved through that portion. Therefore, there is a case where
the intended substance is hard to be collected.
[0083] As the size of substances (particles, molecules) to be
concentrated on the hollow space is small, attention should be paid
to the width of an electrode. Because an area above the electrode
will be a portion which is low in density of electric flux line for
the substance than the hollow space. The reason why is that since a
electric flux line is also generated from an edge of an electrode
in contact with the hollow space, a degree of influence caused by
the electric flux line generated from an edge of an electrode in
contact with the hollow space is different depending on the size of
the substance. Where the substances to be concentrated on the
hollow space are small, this problem can be solved by narrowing the
width of an electrode having the hollow space.
[0084] The shape of the electrode and the hollow space may be a
circle, oval or a polygon, the shape of which is not particularly
restricted. Also, the width of the electrode itself may be wider or
a thin like a wire. In short, the construction of an electrode may
be employed so that an electrode is not present in an area in which
detected objects subjected to the negative dielectrophoretic force
are concentrated, and in a vertical direction thereof.
[0085] Since even the same electrode construction, there appears a
difference in a region where the measured objects are concentrated
due to the change of the frequency of the electric field applied,
and conductivity and dielectric constant of the measured object and
the medium, the electrode construction may be decided according to
the frequency of the electric field applied according to the using
object. Conversely speaking, the substances to be measured can be
concentrated at the desired position by varying the frequency or
the like adjusting to the electrode construction.
[0086] Preferably, the hollow space 12 may be formed in the
electrode, for example, by physical means such as a cutting method
using, for example, a suitable knife or the like and embossing
method, chemical means such as etching for removing an electrode,
for example, using an etching liquid, or for example, by physical
and chemical means such as Reactive Ion Etching (RIE) using a
reactive gas formed into plasma by a high frequency power supply,
and so on.
[0087] The electrode formed with the vacant space 12 of the present
invention is preferably prepared, for example, by the fine
processing technique (Biochim. Bophys. Acta. 964, 231-230 and so
on) as described below:
[0088] (A) For example, a resist is coated on a base plate having
copper, gold, aluminum or the like laminated thereon, and an
electrode photomask is laminated on the resist. Then, light is
irradiated to expose and develop the resist to dissolve a resist
corresponding to a vacant space and a portion other than the
electrode, which is then dipped into an etching liquid to apply
etching to the electrode surface (aluminum surface), and the
remaining resist on the electrode surface is removed. It is noted
that the resist may be a positive resist for removing a portion
exposed to light or a negative resist for removing a portion not
exposed.
[0089] (B) Lift off method
[0090] After a resist is coated on a base plate, an electrode
photomask is laminated on the resist, to which is applied exposure.
Then development is carried out to remove a resist corresponding to
an electrode portion, and an electrode material is laminated on the
whole upper surface by vapor deposition or sputtering. Then, a
resist corresponding to a portion other than the electrode and a
vacant space (an electrode is laminated on the upper surface) is
removed.
[0091] (C) Metal mask method
[0092] A metal mask with only the electrode portion applied with
hollowing is laminated on a base plate, on which upper surface is
coated with an electrode material by vapor deposition or
sputtering. Then, the metal mask (an electrode material is
laminated on the upper surface) is removed.
[0093] In the present invention, an electrode is one made of
conductive materials such as, for example, aluminum, gold, copper
and the like. Its structure can be any structure capable of causing
dielectrophoretic forces, that is, forming a horizontally and
vertically non-uniform electric field, including, for example, an
interdigital shape [J. Phys. D: Appl. Phys. 258, 81-89 (1992);
Biochim. Biophys. Acta., 964, 221-230 (1988), and the like].
[0094] The electrode of the present invention is, preferably,
formed on the upper surface and/or the lower surface of the base
plate(substrate). Normally, since the liquid containing the
substance to be measured is caused to flow above the electrode, an
electrode formed on the upper surface of the base plate is used.
However, an electrode is placed in a state that floated in hollow,
and the liquid containing the substance to be measured can be flown
below the electrode. In this case, an electrode formed on the lower
surface of a base plate or on both upper and lower surface of a
base plate is used.
[0095] The electrodes used in the present invention include, for
example, an electrode in the shape having many electrodes of the
same shape (hexagon) associated, as shown in FIGS. 3 and 4, and an
electrode formed such that a cathode and an anode are provided
internally and externally, respectively, and longitudinal and
lateral parts are made to the same or somewhat different, as shown
in FIG. 5.
[0096] Since in the electrode as shown in FIGS. 3 and 4, negative
dielectrophoretic regions can be formed in not only one place but
several places, several hollow spaces having an area which is low
in density of the same electric flux line can be prepared, whereby
the fluorescent strength of several places is measured and averaged
to thereby obtain data with reliability.
[0097] Further, in an electrode provided with a cathode and an
anode internally and externally, respectively, as shown in FIG. 5,
there is one measuring place, but since a space require is small,
that can be contributed to integration of measurement of many
inspected objects.
[0098] Other concrete examples of electrodes as shown in FIGS. 3
and 4 include a shape in which many triangular outwardly projecting
parts are associated in a spaced relation opposite to upper and
lower portion of a linear web as shown in FIG. 6, a shape in which
many trapezoidal outwardly projecting parts are associated in a
spaced relation opposite to upper and lower portion of a linear web
as shown in FIG. 7, a shape in which many hexagons are associated
linearly as shown in FIG. 8, a shape in which many square outwardly
projecting parts are associated in a spaced relation opposite to
upper and lower portion of a linear web as shown in FIG. 9, and a
shape in which many semicircular outwardly projecting parts are
associated in a spaced relation opposite to upper and lower portion
of a linear web as shown in FIG. 10. While in (A) and (B) in FIGS.
6 to 10, shapes of ends are different, but either of them will
suffice.
[0099] Further, other concrete examples of electrodes as shown in
FIG. 5 include, for example, as shown in FIGS. 11(A) to (G),
electrodes in which an external anode is formed to be polygon such
as square and octagon, circle, semi-circle, and oval; and as an
internal cathode, a cathode head located in a central part of the
cathode is formed to be polygon such as square and octagon, circle
and the like. In the present invention, any electrode can be used
as long as the electrode itself can be used for dielectrophoresis
for forming a hollow space, and the kind of electrodes is not
restricted.
[0100] A base plate (substrate) used when an electrode is prepared
is not particularly restricted if it can be used in this field, and
a base plate formed of a non-conductive material, for example, such
as glass, plastics, quartz, silicon or the like is preferred.
[0101] The base plate may be formed of a transparent material, but
a material need not always be a transparent material if excitation
light is not substantially reflected, or light is permeated to such
an extent as capable of measuring absorbance.
[0102] The electrode may be similar to prior art except formation
of a vacant space, and an organic layer may be formed on the
electrode to prevent adsorption of various materials on the
electrode.
[0103] For manufacturing the electrophoretic apparatus of the
present invention using the electrode of the present invention
formed with the vacant space as described above, those other than
the electrode may be formed in a manner similar to prior art.
[0104] For embodying the separation method of the present invention
using the electrode and the dielectrophoretic apparatus of the
present invention formed with the vacant space as described above,
the separation method itself may be carried out in a manner similar
to prior art.
[0105] Namely, a liquid containing substances to be separated, a
liquid in which for example, two or more kinds of substances
(molecules or particles) are dissolved or suspended is placed in
presence within a non-uniform electric field formed using the
electrode as described above, and separation may be accomplished
due to a difference in the dielectrophoretic force exerting on the
substances. It is noted that an electric field applied in the
present invention may be either DC electric field or AC electric
field, but AC electric field is preferred.
[0106] In the separation method of the present invention, granular
substances of 100 nm to 100 .mu.m are easily concentrated on an
area which is lower in density of electric flux line. Because the
granular substances having the size to some extent may easily
concentrated on an electrode having an area which is low in density
of electric flux line in which substances to be measured are
concentrated in the vacant space and above or below position of the
space. However, it is possible, even when substances to be
separated or measured are small particles or molecules, to
constitute an electrode capable of forming an area which is low in
density of electric flux line in upper and lower directions of the
vacant space by narrowing the width of an electrode or deepening
the depth (the distance between the electrode base plate and the
cover glass and/or the distance from the vessel bottom to the
electrode). In short, since the influence of electric flux line
received by particles is different according to the size of
particles, when the particle having the size to some extent is
applied to the separation method of the present invention, an
electrode in which the particles are concentrated in the vacant
space or in upper and lower directions thereof can be easily
formed.
[0107] Accordingly, for separating molecules or small particles,
which are measured materials, in a solution of molecules or a
suspension of small particles, a complex in which substances to be
measured (through "substances binding to substances to be
measured", if necessary) are bound to substances subjected to
influence by the negative dielectrophoretic force, preferably,
granular substances having the size of 100 nm to 100 .mu.m is
subjected to the separation method using a dielectrophoresis. This
is, because of the fact that if the size of particles is too small,
the width of the electrode need be extremely narrowed.
[0108] The granular substances are bound as described above whereby
the substances are enlarged, and so, separation of the substances
to be measured is facilitated. Accordingly, the granular substances
function as substances for enhancing separation.
[0109] The granular substance used in the present invention
includes inorganic metal oxides such as silica and alumina; metals
such as gold, titanium, iron, and nickel; inorganic metal oxides
and the like having functional groups introduced by silane coupling
process and the like; living things such as various microorganisms
and eukaryotic cells; polysaccharides such as agarose, cellulose,
insoluble dextran; synthetic macromolecular compounds such as
polystyrene latex, styrene-butadiene copolymer,
styrene-methacrylate copolymer, acrolein-ethylene glycol
dimethacrylate copolymer, styrene-styrenesufonate latex,
polyacrylamide, polyglycidyl methacrylate, polyacrolein-coated
particles, crosslinked polyacrylonitrile, acrylic or acrylic ester
copolymer, acrylonitrile-butadiene, vinyl chloride-acrylic ester
and polyvinyl acetate-acrylate; relatively large biological
molecules such as erythrocyte, sugars, nucleic acids, proteins and
lipids, and the like.
[0110] The "granular substance" are normally bound to "substance
binding to substance to be measured" for use. By doing so, it can
be bound to "substance to be measured" in a sample. However, the
granular substance may be bound directly to the substance to be
measured by a chemical binding method, for example, such as a
method for introducing a functional group into the surface of the
granular substance and afterwards binding through the functional
group, or a binding method the granular substance to the substance
to be measured through a linker.
[0111] Further, for binding the granular substance to the
"substance binding to the substance to be measured", a method
similar to a method for labeling the measured substance by a
labeling substance described later may be employed.
[0112] Where a substance having properties capable of specifically
binding to the substance to be measured directly is used as the
granular substance, the operation as described above is
unnecessary. The granular material as described includes, for
example, neucleic acid, protein, lipid and so on.
[0113] The "substance binding to the substance to be measured" used
in the present invention is bound to the granular substance for use
to form a complex of the substance to be measured, the "substance
binding to the substance to be measured", and the granular
substance from the substance to be measured in a sample, and a
complex of a molecule other than the substance to be measured, the
"substance binding to the substance to be measured" and the
granular substance may be not formed substantially, which is not
particularly restricted. In short, even if being bound to the
substances other than the substance to be measured, it will suffice
if that may not form the aforesaid three complex substance.
However, it is actually preferred that the "substance specifically
binding to the substance to be measured is used.
[0114] A "substance binding to the substance to be measured" refers
to a substance binding to the "substance to be measured" by
interactions such as an "antigen"-"antibody" reaction, a "sugar
chain"-"lectin" reaction, "an enzyme"-"inhibitor" reaction, a
"protein"-"peptide chain" reaction, and a "chromosome or nucleotide
chain"--"nucleotide chain" reaction. If one partner is the
substance to be measured in each combination described above, the
other is a "substance binding to the substance to be measured" as
described above.
[0115] For forming a complex of binding the substance to be
measured in a sample with the granular substance directly or
through the "substance binding to the substance to be measured" a
sample containing the substance to be measured, the granular
substance and, if necessary the "substance binding to the substance
to be measured" are, for example respectively dissolved, dispersed
or suspended in water or a buffer liquid, for example, such as tris
(hydroxymethyl amino methane) buffers, a Good's buffer, a phosphate
buffer, borate buffer into a liquid material, and these liquid
material may be mixed and contacted with each another.
[0116] The separation method of the present invention is roughly
divided into two methods as follows:
[0117] [Separation Method 1]
[0118] First, where the substance to be measured, or the complex of
the substance subjected to influence of the negative
dielectrophoretic force (substance for enhancing separation) and
the substance to be measured(through "substance binding to the
substance to be measured", if necessary) exhibits the same negative
dielectrophoretic force as that of the substance other than the
substance to be measured, in case of the substance to be measured
or the complex showing the greater dielectrophoretic force than
that of the substance other than the substance to be measured, only
substantially the substance to be measured, or substance for
enhancing separation and the complex of substance for enhancing
separation and the substance to be measured receive the great
dielectrophoretic force and are separated.
[0119] Namely, for example, by suitably setting the electric field
strength and the medium conditions in such a way that the substance
to be measured or the complex substance of the substance subjected
to influence of the negative dielectropherotic force and the
substance to be measured (through "substance binding to the
substance to be measured, if necessary) is concentrated in the
vacant space above the dielectropherotic electrode or in the upper
and lower directions thereof, but that the substances other than
the substance to be measured are not concentrated, these substance
to be measured and the substance other than the substance to be
measured can be separated.
[0120] The method of the present invention is suited for separation
in the state free from flow. However, the so-called
dielectrophoretic chromatography apparatus (Field Flow
Fractionation apparatus) which carries out separation by the
interaction of the dielectrophoretic force generated in molecules
by the electric field and the movement of molecules, may be used to
carry out separation. In this case, by suitably setting the flow
velocity (speed is made slow) in such a way that only substance to
be measured or the complex of the substance subjected to influence
of the negative dielectrophoretic force and the substance to be
measured (through "substance binding to the substance to be
measured, if necessary) is collected in the vacant space of the
electrode or in the upper and lower directions by the
dielectrophoretic force, these substance to be measured and the
substances other than the substance to be measured can be
separated. In the condition that the substance trapped in the
hollow space of the electrode or in the upper and lower directions
thereof is not moved by the flow, many samples can be applied to
the hollow space of the electrode by the measurement in the flow,
thus enhancing the measurement sensitivity.
[0121] [Separation Method 2]
[0122] Second, where the substance to be measured or the complex of
the substance subjected to influence by the negative
dielectropherotic force and the substance to be measured (through
"substance binding to the substance to be measured", if necessary)
is one subjected to influence by the negative dielectropherotic
force different from substances other than the substance to be
measured, namely where the substance to be measured or the complex
of the substance for enhancing separation (substance subjected to
influence by the negative dielectropherotic force) and the
substance to be measured exhibits the negative dielectropherotic
force and the substances other than the substance to be measured
exhibits the positive dielectropherotic force, either of {circle
over (1)} the substance to be measured or the complex of the
substance to be measured and the substance subjected to influence
by the negative dielectropherotic force and {circle over (2)} the
substances other than the substance to be measured moves to the
hollow space or in the upper and lower directions thereof while the
other moves to a different electrode region whereby the substance
to be measured can be separated from the substances other than the
substance to be measured.
[0123] When the substance to be measured separated by the
separation method according to the present invention can be
detected by a method according to properties own by the substance,
the presence or absence of the substance to be measured contained
in a sample can be measured (detected).
[0124] Namely, using the dielectrode according to the present
invention, the dielectrode constitution and the dielectrophororetic
apparatus, a liquid material (sample) containing the substance
subjected to influence by the negative dielectropherotic force
generated by application of voltage to the electrode [or substance
to be measured or the complex of the substance for enhancing
separation and substance to measured (through "substance binding to
the substance to be measured, if necessary")] is located at the
electrode according to the present invention, or the vacant space
or in the vicinity thereof, or is caused to flow above or below
thereof, whereby the substances subjected to influence by the
negative dielectrophoretic force are concentrated on the vacant
space, above or below thereof, and afterwards, the substance to be
measured in a sample can be detected by optically detecting the
substance.
[0125] The substance to be measured in the above-described method
is that can be measured by any optical method, or that can be
labeled by an optically detectable labeling substance, or bound to
the "substance binding to the substance to be measured" that can be
measured (detected), or that can be labeled by an optically
detectable labeling substance.
[0126] In the present invention, the substance to be measured or
the "substance binding to the substance to be measured" may be
labeled by the optically detectable labeling substance, and
labeling itself may be carried out by a well-known labeling method
generally carried out in a conventional method generally used in
the field of, for example, well-known EIA, RIA, FIA or a
hybridization method.
[0127] The optically detectable labeling substances which can be
used in the present invention are any substances usually used in
the art of enzyme immunoassay (EIA), fluoroimmunoassay (FIA),
hybridization method, and the like, and are not particularly
limited. However, the labeling substance capable of being detected
by the fluorescent strength, the light emission strength or the
absorbance is particularly preferred.
[0128] In the above-described method, as the "substance binding to
the substance to be measured", the "substance binding to the
substance to be measured" that can be measured (detected) by any
optically detectable method or that can be labeled by an optically
detectable labeling substance is generally used.
[0129] More concretely, the detection method according to the
present invention may be carried out in a manner as described
below.
[0130] The substance to be measured or the complex of the substance
to be measured and the separation enhancing substance (if
necessary, through the substance binding to the substance to be
measured and/or the substance binding to the substance to be
measured labeled by the optically detectable labeling substance)
obtained by reacting the substance to be measured and the
separation enhancing substance (if necessary, and the substance
binding to the substance to be measured and/or the substance
binding to the measured substance labeled by the optically
detectable labeling substance) and the substances other than the
substance to be measured (for example, the free substance binding
to the substance to be measured or the free labeled substance to
binding the substance to be measured ) are separated according to
the separation method of the present invention as mentioned above.
Next, the separated substance to be measured or the separated
complex is optically detected on the basis of properties of the
substance to be measured or the substance binding to the substance
to be measured (or the labeling substance binding to the substance
binding to the substance to be measured in the complex) in the
complex to measure the presence or absence of the substance to be
measured in the sample.
[0131] Further, according to the present invention, not only the
presence of the substance to be measured in the sample can be
detected, but also the amount of the substance to be measured in
the sample can be measured quantitatively. The quantitative
measurement of the substance to be measured may be done similarly
to prior art where the complex is not formed, and in case where the
complex substance is formed, the following method may be
employed.
[0132] That is, the substance to be measured or the complex of the
substance to be measured and the separation enhancing substance (if
necessary, through the substance binding to the substance to be
measured and/or the labeled substance binding to the measured
substance) and the substances other than the substance to be
measured [for example, the free substance binding to the substance
to be measured (or the free labeled substance binding to the
substance to be measured )] are separated according to the
separation method of the present invention as described above.
Next, the amount of the separated substance to be measured or the
substance binding to the substance to be measured in the complex
(or the optically detectable labeling substance binding to the
substance binding to the substance to be measured in the complex),
or the amount of the free substance binding to the substance to be
measured (or the optically detectable labeling substance binding to
the free labeled substance binding to the substance to be measured)
are obtained by the optical measurement method according to these
properties, and the amount of the substance to be measured in the
sample can be obtained on the basis of the obtained amount.
[0133] In the above-described method, in order to obtain the amount
of the substance to be measured in the sample on the basis of
obtained amounts of the substance to be measured, the substance
binding to the substance to be measured or the labeling substance,
for example, the quantity of specific molecules in the sample may
be calculated, by using a calibration curve showing a relationship
between the amount of the substance to be measured, and the amount
of the substance binding to the substance to be measured in the
complex (or the labeled substance binding to the substance to be
measured) or the amount of the free substance binding to the
substance to be measured (or the optically detectable labeling
substance in the labeled substance binding to the substance to be
measured ), obtained by carrying out the same measuring method
mentioned above except for using a sample whose concentration of
the substance to be measured is known.
[0134] According to the present invention, the substance to be
measured ( molecules to be measured) can be concentrated in the
hollow space of the electrode or in the upper and lower directions
thereof. When the excitation light is irradiated on the
concentrated measured molecules, since the electrode is not present
under the molecules, the background caused by being reflected even
on the electrode is not detected, as compared with the case using
the conventional electrode, as shown in FIG. 12(A). As a result,
the S/N ratio is enhanced, as compared with prior art and the
measuring sensitivity is enhanced.
[0135] Further, if the electrode of the present invention is used,
since the electrode is not present under the substances to be
measured, a fluorescent detector can be provided on the opposite
side as shown in FIG. 12(B). Further where it is provided on the
opposite side, the S/N ratio is enhanced (slit effect) since the
parts other than the region where the substances to be measured are
concentrated are covered with the electrode, whereby in said parts
the excitation light irradiated from the upper surface does not
reach the lower surface, and therefore, the background can be
reduced.
[0136] Further, according to the present invention, since the
measurement can be done from the lower surface, the absorbance of
the substances to be measured is measured, which has been
heretofore impossible, to enable qaualitative (detection) and
quantitative measurement of the substances to be measured.
[0137] In this case, the S/N ratio is further enhanced (slit
effect) since the parts other than the region where the substances
to be measured are concentrated are covered with the electrode,
whereby in said parts light does not permeate through the electrode
from the upper surface to the lower surface, and therefore, the
background can be further reduced.
[0138] In the following, the invention 2 will be described in
detail.
[0139] FIG. 14 shows an embodiment of the present invention,
showing an example in which an electrode 3 is supported in a
lengthwise spaced relation by a convex member 2 (a support column)
on a substrate(a glass substrate) 1.
[0140] A "lower level place than electrode level" (a communication
groove) 4 which is semicircular in section is formed between the
electrodes 3, 3, as shown in FIG. 14(B), and communication grooves
4, 4 adjacent to each other are communicated at parts other than
the convex member 2, as shown in FIG. 14(A). However,
alternatively, the electrode 3 is supported by a wall (a convex
member) 2', and grooves 4', 4' adjacent to each other are isolated
by the wall 2' so as not to be communicated, as shown in FIG.
15(B).
[0141] In the embodiments shown in FIGS. 14 and 15, portions other
than the convex members 2 and 2' are formed on the "lower level
place than electrode 3 level" (4 and 4').
[0142] However, a concave portion (hole) may be singly or in plural
in a spaced relation provided in a part between the electrodes 3,
3, but preferably, the whole or a major portion between or among
electrodes is formed in a lower level place than the electrode (4
or 4')level as shown in FIGS. 14 and 15 to enhance the collecting
ability.
[0143] Where the concave portion (hole) is formed in a part between
the electrodes 3, 3, preferably, it may be formed in a minimum gap
5 between the electrodes. Since this portion is high in electric
field strength, if the concave portion (whole) is formed in this
portion, the collecting ability is further enhanced. However, if
that is formed in the whole including this portion, further the
collecting ability can be enhanced, because a portion for trapping
molecules increases.
[0144] The width of the groove 4 (the same as the distance between
the electrodes 3, 3 in the case shown in FIGS. 14 and 15) is
suitably decided according to the size of substances as separated
substances by the dielectrophoresis and is said absolutely though
giving great effect to the electric field strength. In the
substance of the size which is micrometer, the width is preferably,
1 time to 100 times of the diameter of the substance, more
preferably, 1 time to 10 times. Further, in case of a bio-molecule
such as a protein, a gene or the like, for example, such as a
peptide, a protein or the like, normally, the width is 1 nm to 10
.mu.m, preferably 1 nm to 5 .mu.m. In case of nucleotide chain
(polynucleotide, oligonucleotide), normally, the width is 1 nm to
100 .mu.m, preferably 1 nm to 50 .mu.m.
[0145] Generally, if the depth is deeper, a portion for trapping a
molecule increases. Further, particularly, in case of Field-Flow
fractionation, the flow velocity at the groove portion is
suppressed to enhance the collecting ability (collecting rate).
However, if being too deep, where it is necessary to measure a
molecule trapped on the electrode by the dielectrophororesis, the
molecule trapped is sometimes hard to be released from the groove
portion or not released. Accordingly, the depth of the groove is,
preferably, {fraction (1/1000)} times to 10 times of the width of
the groove, more preferably, {fraction (1/1000)} times to 1
time.
[0146] With respect to the depth of the groove, if isotropic
etching is used for formation as shown in FIGS. 14 and 15, when the
groove is made more than the width of the electrode, the convex
member which holds the electrode is totally dug away whereby the
electrode 3 is peeled off. Accordingly, when the groove is formed
by this method, the depth of the groove is set to 1/2 or less of
the maximum electrode width.
[0147] Where anisotropic etching of a silicon wafer is used for
formation, as shown in FIG. 15(B), etching progresses only in a
direction of depth at an angle of about 55 degrees. Accordingly,
where etching is made by this method, the maximum distance
depthwise (the distance between electrodes.div.2).times.1.42 (tan
55 degrees) results.
[0148] As shown in FIG. 15(C), where formation is made by RIE or
LIGA, etching progresses substantially vertically. Accordingly,
where etching is made by these methods, the depth of the groove is
in the range described above, namely, preferably, {fraction
(1/1000)} times to 10 times, more preferably {fraction (1/1000)}
times to 1 time.
[0149] The spacing of the groove (=width of the electrode itself)
is not affected by the separated object if limiting to separation
by the positive dielectrophororesis. It is normally from the
processing accuracy in the fine processing technique to 1 nm to 50
.mu.m, more preferably, 1 nm to 10 .mu.m.
[0150] The groove by the isotropic etching shown in FIG. 15(A) is
formed by etching a glass base plate or a plastic base plate. In
the isotropic etching, various shapes are formed according to the
extent of etching such as the case where the electrode 3 is
supported by the wall 2 on the base plate and the grooves 4, 4
adjacent to each other are formed so as to be isolated by the wall
2, or the case where the electrode 3 is supported by the convex
member 2 on the base plate, and the grooves (communication grooves)
4, 4 adjacent to each other are communicated.
[0151] The groove by the anisotropic etching shown in FIG. 15(B) is
formed by etching a silicon base plate. In this case, the electrode
3 is supported on the wall 2' on the base plate, and the grooves
4', 4' adjacent to each other are isolated by the wall 2'.
[0152] The groove by RIE shown in FIG. 15(C) is formed by etching a
silicon or SiO.sub.2 base plate, and the groove by LIGA is formed
by etching polymer, ceramic, plastic base plate etc. In these
cases, the electrode 3 is supported on the wall 2" on the base
plate, and the grooves 4", 4" adjacent to each other are isolated
by the wall 2".
[0153] In the isotropic etching shown in FIGS. 14 and 15(A),
generally, the groove or the communication groove 4 is formed to
have a shape whose section is semicircular, or semi-oval. When a
groove is formed by the anisotropic etching shown in 15(B),
generally, the groove 4' is subjected to etching into a
substantially V-shape finally via a substantially trapezoid in
section. When a groove is formed by RIE or LIGA shown in FIG.
15(C), generally, etching is made to a substantially square in
section. Accordingly, various sectional shapes are formed according
to the way of etching and the way of forming "a lower level place
than electrode level", but in the present invention, the shape of
"a lower level place than electrode level" (such as a communication
groove, a groove, a concave part, etc.) are not particularly
limited.
[0154] A wall or a convex member 2 in FIG. 15(A) is formed into a
shape in which a central part is bound; a wall 2' in FIG. 15(B) is
formed into a trapezoidal shape; and a wall 2" in FIG. 15(C) is
formed into a square shape, but the wall, the convex member 2, the
wall 2', and the wall 2" may be any shape as long as they can
support the electrode 3, and are not particularly limited.
[0155] The electrode 3 used in the present invention is formed of a
conductive material, for example, such as aluminum, gold or the
like, and the construction thereof will suffice to be one which
produce the dielectrophoretic force, that is, a non-uniform
electric field in horizontal and vertical directions, for example,
an interdigital shape [J. Phys. D: Appl. Phys. 258, 81-88, (1992),
Biochim. Biophys. Acta. 964, 221-230, (1988), etc.] being
listed.
[0156] More concretely, preferable are, as shown in FIG. 16, (A) a
shape in which many triangular outwardly projecting parts 7a are
formed in a spaced relation opposite to upper and lower parts of a
linear web-like part 6; (B) a shape in which many square outwardly
projecting parts 7b are formed in a spaced relation opposite to
upper and lower parts of a linear web-like part 6; (C) a shape in
which many trapezoidal outwardly projecting parts 7c are formed in
a spaced relation opposite to upper and lower parts of a linear
web-like part 6; (D) being sine wave shape at upper and lower
portions, a shape in which many sine wave convex parts 8 and
concave parts 9 (concave part 9 and convex part 8) are formed
linearly opposite to upper and lower portions; and (E) being
saw-tooth shape at upper and lower portions, a shape in which many
convex parts 8' of saw-tooth and concave parts 9' (concave part 9'
and convex part 8') are formed linearly opposite to upper and lower
portions. However, any shape can be used if the electrode can be
used for dielectrophoresis, and the shapes are not particularly
limited.
[0157] Such an electrode as described is normally prepared by
providing a pair or more electrodes having shapes as described
above on comb-tooth-wise on a base plate formed of a non-conductive
material, for example, such as glass, plastic, quartz, silicon,
etc. by using known fine processing technique [Bichim. Bioophys.
Acta., 964, 221-230, etc.]. Further, the distance between the
electrodes 3 opposite (adjacent) to each other is not particularly
limited as long as a non-uniform AC electric field of strong
electric field strength can be formed, and should be suitably set
according to the kind of molecules intended.
[0158] The thickness of the electrode 3 may be similar to prior
art, and concretely, the thickness is normally 0.5 nm or more,
preferably, 0.5 nm to 1000 nm, more preferably, 1 nm to 1000
nm.
[0159] The electrode 3 may be similar to prior art except the
thickness, and an organic layer may be formed on the electrode in
order to prevent adsorption of various materials on the
electrode.
[0160] The dielectrophoretic apparatus according to the present
invention may be manufactured in a manner similar to prior art
except "a lower level place than electrode level" (such as a
communication groove 4, a groove 4', a concave portion etc.) such
as a flow path and a dielectrophoretic electrode.
[0161] The "lower level place than electrode level" may be formed,
for example, by excavating a base plate between electrodes by means
of physical means such as an excavating method using a suitable
knife or the like, a LIGA (Lithographile Galvanoformung Abformung)
method using a synchrotron radiant light and an embossing method
using a suitable embossing die; chemical means for excavating a
base plate, for example, using an etching liquid for a base plate;
or physical and chemical means such as etching using reactive gases
formed into plasma by a high frequency power supply [Reactive Ion
Etching (RIE)]. It is noted that the above-described means may be
combined suitably to carry out excavation of a substrate.
[0162] As an etching liquid, a known etching liquid may be selected
according to material of a substrate. Where a lower level place
than electrode level is formed in a part of a substrate, etching
may be accomplished with masking is suitably applied to a portion
which is not desired to be excavated.
[0163] For embodying the separation method of the present invention
using the dielectrophoretic apparatus according to the present
invention, the separation method itself is the same as prior
art.
[0164] That is, a liquid containing a substance to be separated,
for example, a liquid in which more than two kinds of substances
(molecules or particles) are dissolved or suspended is present in a
non-uniform electric field formed using the electrode (electrode
base plate) as described above whereby separation may be
accomplished by a difference of the dielectrophoretic force
exerting on the substances.
[0165] Generally, a non-uniform electric field is formed
horizontally and vertically within a flow path on the substrate to
cause to flow a liquid containing a substance to be separated from
an inlet, and separation may be accomplished by a difference of the
dielectrophoretic force exerting on the substances. However, of
course, the substance may be separated into a component held in a
specific portion of an electrode and a component not held for
carrying out separation without generating a flow.
[0166] For separating by a difference of the dielectrophoretic
force exerting on the substances (molecules, particles), the
substance may be separated into a molecule etc. held in a specific
portion of an electrode and a molecule etc. not held. Or, since
molecules subjected to a stronger dielectrophoretic force move
later than molecules subjected to a weak dielectrophoretic force,
separation may be accomplished making use of the fact that a
difference is produced in moving time.
[0167] As shown by an arrow in FIG. 17, when a liquid containing a
substance to be separated in a direction crossing the lengthwise of
an electrode is caused to flow into a flow path of the apparatus
according to the present invention, the flow velocity in the
communication passage (groove) 4 becomes slower than that of the
flow path portion so that the drag Fv of fluid applied to the
molecule entered the communication groove 4 can be reduced.
Further, by the provision of the communication groove 4 between the
electrodes 3, 3, the range affected by the electric field becomes
widened, and the space where the trapped molecules are stocked
becomes widened whereby the collecting rate (ability) is
enhanced.
[0168] The measuring method of the present invention may be carried
out in conformation with the known method as described above other
than that using the separation method of the present invention, and
the reagents used may be suitably selected from the well-known
reagents.
[0169] While the present invention will be further described
hereinafter concretely with reference to examples and reference
examples, the present invention is not at all limited thereto.
EXAMPLES
Example 1
Preparation of an Electrode of the Present Invention Formed with a
Vacant Space by Etching
[0170] The electrode according to the present invention was
prepared by coating a resist on a glass base plate applied with
aluminum vapor deposition, then exposing through laminating a
photomask having an electrode and vacant space pattern depicted by
an electron beam depicting device on the resist, and developing the
resist, dissolving a resist film corresponding to the vacant space
and portions other than the electrode, and thereafter dipping it
into an etching liquid to apply etching to an aluminum surface, and
removing the resist remained on the aluminum surface to form an
electrode having a vacant space shown in FIG. 13.
[0171] The pattern of the vacant space was changed to prepare
electrodes 1 to 4 different in length (.mu.m) of a) to e) in FIG.
13. Table 1 shows the length (.mu.m) of a) to e) of electrodes 1 to
4 prepared.
1 TABLE 1 Electrode 1 Electrode 2 Electrode 3 Electrode 4 (.mu.m)
(.mu.m) (.mu.m) (.mu.m) a 14 8 8 8 b 8 2 2 2 c 5 5 10 15 d 2 2 2 2
e 3.5 3.5 3.5 3.5
Example 2
Dielectrophoretic Test of Beads on a Hollow Electrode
[0172] Where beads having a diameter of 1 .mu.m was subjected to
dielectrophoresis using a conventional electrode, beads are
concentrated (gathered ) at a position on the electrode whose field
strength is weak. In the design of the electrode prepared in
Example 1, the aluminum electrode portion in a region where the
beads are gathered are excluded.
[0173] A dielectrophoretic test was conducted under the electric
field that the beads show the negative dielectrophoresis on the
electrode (electrode 2 in Table 1) prepared in Example 1, using
beads having a diameter of 1 .mu.m with the fluorescent-labeled
surface thereof.
[0174] A sample solution with the beads suspended was dropped above
the electrode substrate(hollow space), and afterward, a cover glass
was put, and observation was made by an optical microscope.
[0175] As a result of observation of the dielectrophoretic test, it
has been confirmed that the beads were concentrated in the hollow
space (vacant space) of the electrode by the negative
dielectrophoretic force. The beads were concentrated while floating
in the solution above the hollow space (near the cover glass).
Reference Example 1
Manufacture of Dielectrophoretic Electrode Substrate
[0176] A multi-electrode array having a minimum gap of 7 .mu.m, an
electrode pitch of 20 .mu.m, and the number of electrodes of 2016
(1008 pairs) was designed, and a photomask according to the design
was made for manufacturing the electrode as follows.
[0177] On a glass substrate on which aluminum was deposited and to
which a photoresist was applied, an electrode pattern as designed
was drawn on an electron beam drawing machine, and then the
photoresist was developed and the aluminum was etched to make the
photomask.
[0178] The electrode substrate was manufactured according to the
method described in T. Hashimoto, "Illustrative Photofabrication",
Sogo-denshi Publication (1985), as follows.
[0179] The photomask thus made was contacted tightly with the
aluminum-deposited glass substrate to which a photoresist was
applied, and then exposed to the electrode pattern with a mercury
lamp. The electrode substrate was manufactured by developing the
exposed glass substrate for the electrode and etching the aluminum
surface, followed by removing the photoresist remained on the
aluminum surface.
Example 3
Formation of "Lower Level Place than Electrode Level" on a
Substrate by Etching
[0180] As shown in FIG. 18, etching was applied to the glass
substrate 1 of the dielectrophororetic electrode prepared in a
manner described in Reference Example 1 to form a communication
groove 4 in a portion among the electrodes 3 on the glass substrate
1.
[0181] As an etching liquid, sodium fluoride sulfuric acid
(NH.sub.4F 3%, H.sub.2S0.sub.4, H.sub.2O) was used. Sodium fluoride
sulfuric acid has properties to dissolve both glass and aluminum,
but since the speed for etching glass is very quick as compared
with that for etching aluminum, a glass portion other than the
aluminum electrode can be subjected to etching with an aluminum
electrode as a mask.
[0182] It is observed that in case where the thickness of aluminum
of an electrode is 40 nm, when etching to the depth of 3 .mu.m or
more is done, an electrode is bent by a flow of water when the
etching liquid is washed with pure water. However, in case of
thickness of 250 nm, the phenomena that the electrode is bent was
not observed.
[0183] A relationship between an etching time (sec.) and the depth
(.mu.m) of a communication groove formed between electrodes, upon
etching, was measured. The result indicated that the etching time
and the depth of a groove to be formed are in a proportional
relation as shown in FIG. 19. The depth of a groove was measured by
cutting an electrode with a glass cutter and observing its section
with a microscope.
Reference Example 2
Manufacturing an Electrode Substrate Having a Glow Path
[0184] In order to separate molecules by the movement of the
molecules under an non-uniform electric field, a flow path on the
electrode substrate manufactured in Example 3 was made using
silicone rubber.
[0185] The silicone-rubber flow path for sending a solution
containing dissolved molecule on the electrode had a depth of 25
.mu.m and a width of 400 .mu.m and was designed such that the flow
path runs through a region in which the electrode on the electrode
substrate was placed.
[0186] Its manufacturing was carried out according to the method
described in T. Hashimoto, "Illustrative Photofabrication",
Sogo-denshi Publication (1985). At first, a sheet-type negative
photoresist having a thickness of 25 .mu.m was applied onto the
glass substrate, exposed through a photomask designed for making
the flow path, and the negative photoresist was developed. Uncured
silicone rubber was cast using the negative-photoresist substrate
as a template, and then was cured to produce a silicon rubber
surface having the concave surface with a height of 25 .mu.m in the
region where the electrode was placed.
[0187] The electrode substrate and the silicone-rubber flow path
were adhered with a two-fluid-type curing silicone rubber such that
the concave surface of the silicone rubber was faced to the region
where the electrode on the electrode substrate was placed. A
syringe for injecting a solution was placed upstream of the flow
path, and an apparatus allowing a solution in which the molecules
were dissolved to flow on the electrode was added to the electrode
substrate.
Example 4
Measurement of Collecting Rate with Respect to Bovine-Serum Albumin
(BSA) Protein
[0188] An electrode formed with a communication groove having the
depth of 2 .mu.m or 4 .mu.m was prepared as in Example 3, a flow
path was prepared as in Reference Example 2, a dielectrophoretic
chromatography device of the present invention was prepared, and
the collecting rate of the device was measured in the following
manner. For the purpose of comparison, with respect to the
dielectrophoretic chromatography device prepared similarly except
that a communication groove is not formed, the collecting rate was
also measured.
[0189] (Sample)
[0190] As a sample, a solution containing FITC labeled BSA
(molecular weight: approximately 65 kD) (60 .mu.g/ml )was used.
[0191] (Operation)
[0192] For preventing adsorption of protein molecules to the
electrode substrate or flow path, a block A (manufactured by Snow
Brand Milk Products CO., Ltd.) was used to block the surface of the
flow path, after which FITC labeled BSA was applied to the
dielectrophoretic chromatography device.
[0193] The average speed of the sample used was 556 .mu.m/sec., and
the electric field was applied for 30 to 120 seconds from a start
of measurement. The collecting rate was measured with respect to
the electric field strength applied at that time of 2.14 Mv/m, 2.5
Mv/m, and 2.86 Mv/m.
[0194] The measurement of the collecting rate was obtained by the
following Equation.
Collecting rate
(%)=[(I.sub.0-I.sub.min).times.100]/(I.sub.0-I.sub.back)
[0195] Wherein I.sub.0represents the fixed value of the fluorescent
strength before application of electric field, I.sub.min represents
the minimum value of the fluorescent strength during application of
electric field, and I.sub.back represents the background.
[0196] (Results)
[0197] FIG. 20 shows the results. In FIG. 20, there is shown the
results obtained by the use of the dielectrophoretic chromatography
device of -.DELTA.- (depth 4 .mu.m), -.quadrature.- (depth 2
.mu.m), and -.diamond.- (depth 0 .mu.m).
[0198] As is clear from the results shown in FIG. 20, the deeper
the depth of groove, the collecting rate (%) enhances. In 2.86
Mv/m, the collecting rate of the apparatus of the present invention
having the communication groove of 4 .mu.m is 40% as compared with
the collecting rate 28% of the conventional apparatus having no
communication groove, and the collecting rate was enhanced by about
43%, in other words, the collecting ability of the substances
intended is remarkably enhanced by the use of the apparatus
according to the present invention.
Example 5
Measurement of Collecting Rate to 500 bpDNA
[0199] 500 bpDNA labeled by intercalator fluorescent dye YOYO-1
(Molecular Probe Ltd.) was used as a sample. The collecting rate
(%) was measured by the dielectrophophoretic chromatography device
of the depth of groove, 0 .mu.m, 2 .mu.m and 4 .mu.m. FIG. 21 shows
the results.
[0200] In FIG. 21, there is shown the results obtained by the use
of the dielectrophororetic chromatography device having the
communication groove of -.DELTA.- (depth 4 .mu.m), -.quadrature.-
(depth 2 .mu.m), and -.diamond.- (depth 0 .mu.m).
[0201] As is clear from the results shown in FIG. 21, Also in this
case, in the electric field strength of 1.5 Mv/m or more, the
collecting rate of the apparatus of the present invention having
the communication groove of depth 4 .mu.m was enhanced by about 20%
as compared with the conventional apparatus having no communication
groove.
[0202] Advantageous Effect of the Invention
[0203] According to the invention 1, since the substances to be
measured can be concentrated (gathered ) in the hollow space of the
electrode or in the upper and lower directions thereof, the
electrode is not present under the substances to be measured, and
therefore, where the fluorescent strength is detected, the
reflection of the excitation light by the electrode under the
measured substances is avoided. As a result, the background is
reduced, the S/N ratio is enhanced, and the measurement sensitivity
is enhanced. Further, the measurement can be made from the lower
surface of the electrode. Further, according to the present
invention, since the measurement can be made from the lower
surface, it is possible to measure the substances to be measured by
the absorbance that has been impossible in prior art.
[0204] When the measurement is made from the lower surface of the
electrode, since the parts other than the region where the
substances to be measured are concentrated are covered with the
electrode, whereby in said parts the excitation light irradiated
from the upper surface does not reach the lower surface, the
background is reduced, the S/N ratio is enhanced and the
measurement sensitivity is enhanced (slit effect). This is an
extremely great advantage.
[0205] According to the invention 2, the provision of lower level
places than electrode level between or among electrodes which has
not at all been done in prior art leads to the remarkable
enhancement of the collecting ability(rate) which has a very
important role for separation of substances by the
dielectrophoresis, which is an enormous effect. This is therefore
an extremely epoch-making invention.
* * * * *