Source: http://www.patentsencyclopedia.com/app/20110259746
Timestamp: 2016-08-30 00:50:53
Document Index: 102674234

Matched Legal Cases: ['art 13', 'arts 7', 'art 6', 'arts 7', 'art 6', 'arts 7', 'art 6', 'arts 8', 'arts 9', 'art 9', 'art 8', 'arts 8', 'arts 9', 'art 9', 'art 8']

[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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
[0036] 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.
[0037] 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
[0038] For achieving the aforementioned objects, the present inventors
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.
[0039] 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.
[0040] 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.
[0041] 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
[0042] 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.
[0043] 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
[0044] 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
[0045] In the following, a case where a positive dielectrophoretic force
exerts on a molecule will be described.
[0046] 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.
[0047] 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 Fd, the force Fv generated by the
drag due to the flow in the flow path, and the force Fth due to the
thermal movement. {circle around (1)} in the case of
Fd>>Fv+Fth, molecules are captured (trapped) on the
electrode, {circle around (2)} in the case of
Fd<<Fv+Fth, molecules are eluted out with flow in
the flow path, regardless of the electric field. {circle around (3)} in
the case of Fd≈Fv+Fth, 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.
[0048] 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 around (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".
[0049] The above and other objects and advantages of the invention will
become more apparent from the following description.
[0050] FIG. 1 is an explanatory view of the negative dielectrophoresis.
[0051] FIG. 2 is a view showing the principle of the positive
[0052] FIG. 3 is a plan view showing an embodiment of an electrode of the
[0053] FIG. 4 is a plan view showing a further embodiment of an electrode
[0054] FIG. 5 is a plan view showing another embodiment of an electrode of
[0055] FIGS. 6A and 6B are plan views showing examples of a conventional
[0056] FIGS. 7A and 7B are plan views showing further examples of a
[0057] FIGS. 8A and 8B are plan views showing other examples of a
[0058] FIGS. 9A and 9B are plan views showing still other examples of a
[0059] FIGS. 10A and 10B are plan views showing other examples of a
[0060] FIGS. 11A through 11G are plan views showing still other examples
of a conventional electrode.
[0061] 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
[0062] FIG. 13 is a plan view showing an electrode of the present
invention prepared in Example 1.
[0063] FIG. 14 are respectively, a plan view (A) and a sectional view (B)
showing a further embodiment of the present invention.
[0064] 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),
[0065] FIGS. 16A through 16E are plan views showing electrodes used in the
[0066] FIG. 17 is a sectional view of a dielectrophoretic chromatography
[0067] FIG. 18 is a sectional view showing an example of forming "lower
level place than electrode level" on a base plate (substrate) according
[0068] FIG. 19 is a graph showing a relationship between etching time and
the depth of a groove measured in Example 3.
[0069] 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.
[0070] 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.
[0071] The preferred embodiments of the present invention will be
[0072] First, the invention 1 will be described in detail hereinafter.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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: [0083] (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. [0084] (B) Lift off method [0085] 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.
[0086] (C) Metal mask method [0087] 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.
[0088] 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].
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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
[0094] 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 elect-rode itself can be used for
dielectrophoresis for forming a hollow space, and the kind of electrodes
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] In the separation method of the present invention, granular
substances of 100 nm to 100 μm 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.
[0102] 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 μm 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
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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"
[0110] 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
[0111] The separation method of the present invention is roughly divided
into two methods as follows:
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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 around (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 around (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.
[0116] 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).
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] More concretely, the detection method according to the present
invention may be carried out in a manner as described below.
[0123] 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.
[0124] 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
[0125] That is, the substance to be measured or the complex of the
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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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
[0130] 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.
[0131] In the following, the invention 2 will be described in detail.
[0132] 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.
[0133] 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.
[0134] 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').
[0135] 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.
[0136] 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.
[0137] 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 μm, preferably 1 nm to 5 μm. In case of nucleotide chain
(polynucleotide, oligonucleotide), normally, the width is 1 nm to 100
μm, preferably 1 nm to 50 μm.
[0138] 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, 1/1000 times to 10 times of the
width of the groove, more preferably, 1/1000 times to 1 time.
[0139] 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.
[0140] 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/2)×1.42 (tan 55 degrees) results.
[0141] 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, 1/1000 times to 10 times, more
preferably 1/1000 times to 1 time.
[0142] 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 μm, more preferably, 1
[0143] The groove by the isotropic etching shown in FIG. (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.
[0144] 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'.
[0145] The groove by RIE shown in FIG. 15(C) is formed by etching a
silicon or Si02 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''.
[0146] 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.
[0147] 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
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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)].
[0155] It is noted that the above-described means may be combined suitably
to carry out excavation of a substrate.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
Preparation of an Electrode of the Present Invention Formed with a Vacant
Space by Etching
[0164] 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. The
pattern of the vacant space was changed to prepare electrodes 1 to 4
different in length (μm) of a) to e) in FIG. 13. Table 1 shows the
length (μm) of a) to e) of electrodes 1 to 4 prepared.
[0165] Where beads having a diameter of 1 μm 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.
[0166] 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 μm with the fluorescent-labeled surface thereof.
[0167] 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.
[0168] 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).
[0169] A multi-electrode array having a minimum gap of 7 μm, an
electrode pitch of 20 μm, 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.
[0170] 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.
[0171] The electrode substrate was manufactured according to the method
described in T. Hashimoto, "Illustrative Photofabrication", Sogo-denshi
Publication (1985), as follows.
[0172] 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.
Formation of "Lower Level Place than Electrode Level" on a Substrate by
[0173] 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.
[0174] As an etching liquid, sodium fluoride sulfuric acid (NH4F 3%,
H2SO4, H20) 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.
[0175] It is observed that in case where the thickness of aluminum of an
electrode is 40 nm, when etching to the depth of 3 μm 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.
[0176] A relationship between an etching time (sec.) and the depth (μm)
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.
[0177] 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.
[0178] The silicone-rubber flow path for sending a solution containing
dissolved molecule on the electrode had a depth of 25 μm and a width
of 400 μm and was designed such that the flow path runs through a
region in which the electrode on the electrode substrate was placed.
[0179] 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 μm 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 μm in the region where the electrode was placed.
[0180] 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.
Measurement of Collecting Rate with Respect to Bovine-Serum Albumin (BSA)
[0181] An electrode formed with a communication groove having the depth of
2 μm or 4 μm 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
[0182] As a sample, a solution containing FITC labeled BSA (molecular
weight: approximately 65 kD) (60 μg/ml)was used.
[0183] 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
[0184] The average speed of the sample used was 556 μm/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.
[0185] The measurement of the collecting rate was obtained by the
Collecting rate (%)=[(I0-Imin)×100]/(I0-Iback)
Wherein I0 represents the fixed value of the fluorescent strength
before application of electric field, Imin represents the minimum
value of the fluorescent strength during application of electric field,
and Iback represents the background.
[0186] FIG. 20 shows the results. In FIG. 20, there is shown the results
obtained by the use of the dielectrophoretic chromatography device of
-Δ- (depth 4 μm), -quadrature- (depth 2 μm), and
-⋄- (depth 0 μm).
[0187] 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 μm 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
[0188] 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
μm, 2 μm and 4 μm. FIG. 21 shows the results.
[0189] In FIG. 21, there is shown the results obtained by the use of the
dielectrophororetic chromatography device having the communication groove
of -Δ- (depth 4 μm), -quadrature- (depth 2 μm), and
[0190] 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 μm was enhanced by about 20% as compared with the
conventional apparatus having no communication groove.
[0191] 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.
[0192] 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.
[0193] 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.
Patent applications by Masao Washizu, Bunkyo-Ku JP
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