JP4779468B2 - Interaction detecting unit, bioassay substrate, and method related thereto - Google Patents

Description

  The present invention relates to an interaction detection unit in which a working electrode is formed edge-free. That is, it belongs to a technical field related to a substrate for bioassay such as a DNA chip (DNA microarray), and particularly relates to a technique for detecting an interaction between substances (hybridization, etc.) using an electrodynamic effect. To do.

  In recent years, DNA chips or DNA microarrays (hereinafter referred to as “DNA chips” in the present application) have been put into practical use. A DNA chip is obtained by collecting and fixing various and many DNA probes as nucleic acids for detection on a substrate surface. Using a DNA chip, it is possible to comprehensively analyze gene expression in cells / tissues by measuring the hybridization between nucleic acids for detection on the surface of the DNA chip substrate and target nucleic acids collected from cells / tissues. it can.

Nucleic acid molecules are known to stretch or move when subjected to the action of an electric field in the liquid phase.
The principle is estimated as follows. In the nucleic acid solution, phosphate ions (negative charge) in the nucleic acid molecule and hydrogen ions (positive charge) in the water molecule form an ion cloud, and a polarization vector (dipole) is generated by the negative charge and positive charge. When a high frequency high voltage is applied, the polarization vector is directed in one direction as a whole, and as a result, the nucleic acid molecule is expanded.
In addition, it is known that when a non-uniform electric field is applied and electric lines of force concentrate on a part, the nucleic acid molecules move toward a site where electric lines of force concentrate (Non-patent Document 1).

In addition, an electrodynamic action called “dielectrophoresis” is known for nucleic acid molecules. The outline is as follows.
When the gap (interval) between the working electrode and the reference electrode is set to several tens to several hundreds of μm and a high frequency electric field of about 1 MV / m or 1 MHz is applied to the DNA solution, dielectric polarization occurs in the DNA. It is stretched linearly parallel to the electric field. By this “dielectrophoresis”, DNA is attracted to the end of the electrode and fixed in a form in which one end is in contact with the electrode (Non-patent Document 2).

Regarding the electrodynamic characteristics of nucleic acids as described above, application to DNA chips is being studied. For example, Patent Document 1 describes a technique for separating complementary strand DNA and non-complementary strand DNA by fixing a DNA probe to electrodes and applying a DC voltage between the electrodes.
JP 2002-168864 A Seiichi Suzuki, et al: “Quantitative analysis on electrostatic orientation of DNA in stationary AC electric field using fluorescence anisotropy”, IEEE transaction on Industrial Applications, Vol.34, No.1, P75-83 (1998) Masao Awazu, “DNA Handling while Watching”, Visualization Information Vol.20 No.76 (2000)

  When an edge portion (corner portion) exists in the working electrode formed in each well (reaction field) of the DNA chip, electric lines of force concentrate on that portion. Therefore, when an electric field is formed, the problem that detection nucleic acids, target nucleic acids, and the like are concentrated on the edge portion of the working electrode, that is, it is difficult to uniformly distribute these substances throughout the working electrode. was there.

  Therefore, the main object of the present invention is to provide means for forming a highly uniform electric field in the reaction field of the interaction between the detection substance and the target substance.

  In the present invention, there is provided an interaction detection unit in which at least a working electrode is formed in an edge-free manner in a predetermined region of a substrate surface facing a reaction field of an interaction between a detection substance and a target substance.

  By forming the working electrode in an edge-free manner in a predetermined region of the substrate surface, it is possible to form a highly uniform electric field in the reaction field when forming an electric field using the working electrode and the reference electrode. Therefore, the nucleic acid for detection, the target nucleic acid, etc. can be distributed almost uniformly throughout the working electrode.

  That is, in both (1) the procedure for fixing the detection substance to the working electrode and (2) the procedure for causing the detection substance fixed on the working electrode and the target substance to interact, the detection substance or the target The substance can be dielectrophoresed substantially uniformly toward the working electrode.

  Therefore, at the stage of fixing the detection substance to the working electrode, the detection substance can be fixed almost uniformly to the entire working electrode, and at the stage of interaction (hybridization etc.), uneven distribution of the target substance is caused. It can be reduced and the accuracy of the interaction can be increased.

  Hereinafter, technical terms according to the present invention will be defined.

  “Edge-free” means that the edge (corner) portion of the electrode does not protrude from the surrounding substrate surface.

  A “detection substance” is a substance such as a biological substance that exists in a medium stored or held in a reaction field and functions as a detector for detecting a substance that specifically interacts with the substance. It is fixed or released on the substrate surface facing the reaction field. A typical example is a nucleic acid (nucleotide chain) such as a DNA probe.

  The “target substance” is a substance such as a biological substance that specifically interacts with the detection substance. A typical example is a nucleic acid (nucleotide chain) having a base sequence complementary to a detection nucleic acid such as a DNA probe and exhibiting hybridization.

  “Interaction” broadly means non-covalent bonds, covalent bonds, chemical bonds or dissociations including hydrogen bonds between substances, for example, hybridization that is a complementary bond between nucleic acids (nucleotide strands), polymer-high Widely includes molecules, macromolecule-small molecules, small molecule-small molecule specific bonds or associations.

  “Bioassay substrate” means a substrate for detecting the interaction by causing an interaction between substances to proceed in a predetermined reaction region on the substrate, and widely includes regardless of the type of the substance, The detection principle of the interaction is not limited.

  “Dielectrophoresis” is a phenomenon in which molecules are driven toward a stronger electric field in a field where the electric field is not uniform. When an AC voltage is applied, the polarity of the polarity is also reversed as the polarity of the applied voltage is reversed, so that the driving effect can be obtained in the same way as in the case of DC (supervised by Teru Hayashi, “Micromachine and Material Technology (CMC Publishing ) ", P37-P46, Chapter 5, Cell and DNA manipulation). In particular, in a high-frequency alternating electric field, force acts on the dipole in proportion to the gradient of the square of the temporal average electric field, causing migration.

  According to the present invention, a highly uniform electric field can be formed in the reaction field of the interaction between the detection substance and the target substance. Therefore, the detection substance or the target substance can be dielectrophoresed substantially uniformly toward the working electrode.

  The present invention will be described below with reference to the accompanying drawings.

  FIG. 1 is a schematic cross-sectional view showing an example of an interaction detection unit A according to the present invention.

  In FIG. 1, a reaction field 2 of interaction between a detection substance and a target substance is formed on a substrate 1 in a well shape (concave shape), and acts on a predetermined region of a substrate surface 11 facing the reaction field 2. The electrode W is formed edge-free. Then, an electric field F is formed in the reaction field 2 using the working electrode W and the reference electrode R, and the detection substance 3 and the target substance (not shown, the same applies hereinafter) are dielectrophoresed toward the working electrode W. Reference sign V indicates a power source for performing dielectrophoresis, and reference sign S indicates a switch thereof.

As described above, by forming the working electrode W on the substrate surface 11 in an edge-free manner, a highly uniform electric field can be formed in the reaction field 2, so that the detection substance 3 or the target substance can be used as the entire working electrode W. Can be distributed almost uniformly.
Therefore, at the stage of fixing the detection substance to the working electrode, the detection substance 3 (reference numeral 3) is dielectrophoresed toward the working electrode W, thereby fixing the detection substance 3 to the entire working electrode W almost uniformly. be able to.
Further, at the stage of detecting the interaction (hybridization etc.), the target substance is dielectrophoresed toward the working electrode W (the detection substance 3 is fixed), so that the target substance is almost near the working electrode W. Since it can be uniformly distributed, detection unevenness can be reduced and the detection accuracy of the interaction can be increased.

The material of the substrate 1 is not particularly limited, but a material that can transmit excitation light (for example, fluorescence excitation light) having a predetermined wavelength is preferable from the viewpoint of being able to excite from the substrate side when detecting an interaction.
That is, for example, a substrate similar to a known optical information recording medium such as CD (compact disc), DVD (digital versatile disc), MD (mini disc) can be used, and glass such as quartz, polycarbonate, polystyrene, etc. These materials are preferred.

  As described above, the reaction field 2 is an interaction field between the detection substance and the target substance. A medium (nucleic acid solution, protein solution, buffer solution, etc.) is stored in the reaction field 2 according to the purpose.

The working electrode W is an electrode that forms an electric field F with the reference electrode R, and is formed edge-free in a predetermined region of the substrate surface 11.
When performing dielectrophoresis, since the working electrode W needs to be formed smaller than the reference electrode R, the working electrode W needs to be formed not on the entire surface of the substrate 11 but on a predetermined region. At that time, by preventing the edge portion of the working electrode W from protruding from the substrate surface 11 (that is, by forming the working electrode W in an edge-free manner), the uniformity of the electric field can be improved.

  The method of forming the working electrode W in an edge-free manner is not particularly limited. For example, a method of digging the substrate surface 11 by etching or the like and embedding an electrode material therein can be used. In addition, as a method of embedding the electrode material, for example, a method of etching back after forming a certain amount of metal (sputtering, CVD, plating, etc.), a method of polishing after forming a certain amount of metal, etc. Can be applied.

  Known electrode materials can be used for the working electrode W and the reference electrode R, and are not particularly limited. For example, metals such as aluminum, gold, and platinum, oxides such as ITO (indium-tin-oxide), silicon whose conductivity is increased by high concentration doping, and the like can be applied. It is preferable to form the working electrode W from a transparent material such as ITO from the viewpoint of being able to excite from the substrate side when detecting an interaction.

  A thin insulating layer (not shown) may be provided on the surface of the working electrode W and / or the reference electrode R. As the material of the insulating layer, for example, SiO2, SiC, SiN, SiOC, SiOF, TiO2 and the like are suitable. By providing an insulating layer on the surface of each electrode, an electrochemical reaction due to an ionic solution stored in the reaction field 2 can be prevented. Moreover, corrosion of the electrode can be prevented by preventing electron transfer between the electrode and the solvent.

The working electrode W may be subjected to a surface treatment so that the detection substance (reference numeral 3) can be fixed by chemical bonding.
As a surface treatment means for the working electrode W, for example, there is a method in which the working electrode W is previously surface treated with an amino group-containing silane coupling agent solution, a polylysine solution, or the like. When surface treatment is performed on a synthetic resin substrate, the surface may be treated with an amino group-containing silane coupling agent solution after plasma treatment and DUV (Deep UV, far infrared) irradiation treatment.
The surface of the working electrode W may be coated with a substance having a functional group such as an amino group, a thiol group, or a carbaxyl group, cysteamine, streptavidin, or the like. When surface-treated with streptavidin, it is suitable for immobilizing biotinylated DNA probe ends. When the surface is treated with a thiol group (SH) group, it is suitable for immobilization of a detection substance D such as a probe DNA having a thiol group modified at the terminal by a disulfide bond (-SS-bond).

In the present invention, it is only necessary that the working electrode W is formed in an edge-free manner, and the working electrode W and the reference electrode R are not limited by the position and configuration.
For example, when the reference electrode R is formed on the well-shaped side wall surface 12 (see FIG. 2), the reference electrode R is arranged on the same substrate surface 11 as the working electrode (see FIG. 3), etc. Included in the present invention. In this case, as shown in FIG. 2 or 3, it is more preferable to form not only the working electrode W but also the reference electrode R in an edge-free manner from the viewpoint of forming a highly uniform electric field.

  FIG. 4 is a cross-sectional view and an upper schematic view showing another example of the interaction detection unit A according to the present invention.

  In FIG. 4 as well as FIG. 1 and the like, the reaction field 2 of the interaction between the detection substance and the target substance is formed in a well shape (concave shape) on the substrate 1, and the substrate surface 11 facing the reaction field 2. In addition, the working electrode W is formed edge-free. Then, an electric field is formed in the reaction field 2 using the working electrode W and the reference electrode R, and the detection substance 3 and the target substance are dielectrophoresed toward the working electrode W. In addition, the code | symbol V shows the power supply at the time of performing dielectrophoresis similarly to FIG. 1 etc., and the code | symbol S shows the switch.

In FIG. 4, as in FIG. 3, the working electrode W and the reference electrode R are arranged side by side on the same substrate surface 11.
For example, when the working electrode W and the reference electrode R are vertically opposed as shown in FIG. 1, in order to increase the capacity of the reaction field 2, it is necessary to increase the distance between the electrodes. Therefore, when the capacity of the reaction field 2 is increased, electrode formation becomes difficult.
On the other hand, when the capacity of the reaction field is reduced, the reaction field 2 is easily dried and the concentration of the solution in the reaction field 2 needs to be relatively high, so that the rate of mismatch complementary strand formation increases.
On the other hand, by arranging the working electrode W and the reference electrode R on the same substrate surface 11, the capacity of the reaction field 2 can be increased, and the distance between the working electrode W and the reference electrode R needs to be increased. There is no.
Therefore, increasing the amount of the solvent in the reaction field 2 can suppress mismatched complementary strand formation, and can prevent the substrate from drying. In addition, since electrode formation becomes easy and the substrate configuration can be simplified, there is an advantage that manufacturing costs can be suppressed.

  In FIG. 4, the reference electrode R is formed in a substantially ring shape at a position surrounding the working electrode W. Thereby, the uniformity of the electric field in the reaction field 2 is securable.

  In addition, in FIG. 4, a discontinuous portion R <b> 1 is provided in a part of the substantially ring shape of the reference electrode R, and the wiring 4 connected to the working electrode W is laid in this portion. Thereby, since the wiring 4 can be formed in the board | substrate surface 11, board | substrate manufacture can be simplified and manufacturing cost can be reduced. In particular, there is also an advantage that the substrate configuration can be simplified because the wiring 4 of the substrate does not interfere with the optical path even when excited from the lower side of the substrate, for example, when detecting an interaction using excitation light.

  In Example 1, the potential distribution when the working electrode was formed edge-free was simulated by the finite element method (FEM).

  FIG. 5 is a diagram showing an electrode structure used in this simulation.

  In the drawing, the schematic diagram on the left is an example of an actual electrode structure. In this figure, the working electrode is formed at the center position and the reference electrode is formed at a position surrounding the working electrode, as in FIG. A discontinuous portion is provided in a part of the substantially ring shape of the reference electrode, and a wiring connected to the working electrode is laid in the portion. Moreover, the reference electrode is also provided with a wiring connected to the external electric wire.

On the other hand, the schematic diagram on the right side of the figure is a schematic diagram showing the electrode structure used in the simulation.
For example, in the actual electrode structure, when the electrodes are arranged as shown in the schematic diagram on the left side, the width of the wiring is sufficiently smaller than the width of both electrodes, and the electric field in the vicinity of the wiring is sufficient as compared with the entire electrode structure. Therefore, even if the wiring is ignored, it can be estimated that there is almost no influence on the potential distribution.
Therefore, in this example, in order to simplify the simulation, the wiring was ignored and the simulation was performed on the assumption that the electrode structure is a structure as shown in the schematic diagram on the right side of FIG.

  FIG. 6 is a diagram illustrating parameters used in the simulation.

  In the drawing, the diagram on the left schematically shows the configuration of the interaction detection unit, and the diagram on the right shows each parameter. Also, the left figure schematically shows a conventional interaction detector (the electrode has an edge part), and the right figure shows the interaction detector according to the present invention (the electrode is formed edge-free). Schematically).

In both figures, the part corresponding to the reference numeral 1 is the electrolyte solution, the part corresponding to the reference numeral 2 is the working electrode, the part corresponding to the reference numeral 3 is the reference electrode, the part corresponding to the reference numeral 4 is the substrate, and the reference numeral 5 is the reference numeral. Corresponding portions respectively indicate the outer walls of the interaction detection unit.
In both figures, when 360 ° is rotated about the Z axis, the configuration is almost the same as that of the interaction detection unit illustrated in FIG.

  In both figures, parameter “a” is the working electrode diameter, parameter “d” is the electrode thickness, parameter “g” is the gap between electrodes (distance between both electrodes), and parameter “w” is The widths of the reference electrodes are shown respectively.

With respect to the electrode structure model shown in the schematic diagram on the right side of FIG. 5, the three-dimensional simulation is dropped into the simulation in the two-dimensional plane of the same radial direction R and the vertical direction Z using the rotation target property, and the finite element method is used. A simulation was performed.
In this simulation, the value of the outer diameter of the interaction detection unit is fixed to 100 μm (radius 50 μm), the value of the working electrode diameter a is fixed to 10 μm (radius 5 μm), the electrode thickness d, and the gap g between the electrodes A finite element method model was created by varying three values of the width w of the reference electrode.
In this simulation, a working electrode and a reference electrode are formed of gold (Au, conductivity σ = 4.5 × 10 ⁷ S / m) on a substrate (SiO ₂ substrate, dielectric constant ε = 3.9), It was carried out on the assumption that a NaCl solution (dielectric constant ε = 80, conductivity σ = 0.981 S / m) as an electrolyte solution was filled in the upper part.
The applied AC voltage was 2 V in amplitude and 1 MHz in frequency. In addition, since the electric field of water may occur with a potential difference of 1.23V and the redox potential of gold is 1.83V, in the case of this AC voltage amplitude, an electrode reaction theoretically occurs. There is a possibility, but since the frequency of the AC voltage is high, the influence is almost negligible.

  The simulation results are shown in FIGS.

FIG. 7 is a diagram illustrating a simulation result of the electric field distribution.
This simulation was performed assuming that the working electrode diameter a was 10 μm (radius 5 μm), the gap g between the electrodes was 10 μm, the reference electrode width w was 50 μm, and the electrode thickness d was 1 μm.

In the figure, the upper two figures are schematic views showing the electrode structure. In these drawings, the cross section of the interaction detection unit is described with the orientation changed in the vertical direction. Further, since this simulation model is subject to rotation, only half of the interaction detection unit is shown in these drawings.
The figure on the left (the figure described as “(a) the conventional method”) shows the electrode structure in the case where the corner portion is present in the electrode as in the conventional case, and the figure on the right (“(b) the invention (edgeless)” ) Schematically shows an edge-free electrode structure.
That is, in the left figure, the working electrode and the reference electrode are provided on the substrate (SiO ₂ ) with corner portions, and the upper side (right direction in the figure) is filled with the electrolyte solution. In the figure, the substrate (SiO ₂ ) is also present in the region between the two electrodes and is in an edge-free state (a state in which both electrodes are embedded in the substrate).

  On the other hand, the lower two figures show the simulation results of the electric field distribution. Similar to the upper figure, the left figure (the figure described as “(a) conventional method”) shows the electrode structure when the electrode has a corner portion, and the right figure (“(b) the present invention”). (Edge-less drawing) schematically shows an edge-free electrode structure. Each region in both figures corresponds to the upper two drawings.

In the lower left figure, the electric field is concentrated at the corners of the working electrode and the reference electrode, whereas in the lower right figure, the working electrode and the reference electrode are embedded in the substrate and formed edge-free. By doing so, the concentration of the electric field can be relaxed.
Therefore, this simulation result suggests that the concentration of the electric field can be prevented and the electric field can be uniformly distributed by forming both electrodes in an edge-free manner.

FIG. 8 is a simulation result in which the electric field distribution in the case where the electrode has a corner portion is numerically divided into the same diameter direction and the vertical direction.
In the figure, the horizontal axis (“Position”) indicates a distance (unit: μm) from a predetermined position, and the vertical axis (“Electric Field”) indicates an electric field (unit: V / m).

The simulation of the diameter a of the working electrode 10 [mu] m (radius 5 [mu] m), 10 [mu] m gaps g between the electrodes, 50 [mu] m width w of the reference electrode, the thickness of the electrode d assuming 5 [mu] m, and, in the same radial electric field _{E r} And by plotting the electric field E _{z in the} vertical direction.

In the case of a plot of the electric field _{Er in} the same radial direction, the horizontal axis indicates the distance from the center of the interaction detection unit. That is, the portion of 0 to 5 μm on the horizontal axis represents the working electrode portion, the portion of 5 to 15 μm represents the gap portion between the electrodes, and the portion of 15 μm or more represents the reference electrode portion.

On the other hand, when the vertical direction of the electric field E _z, the horizontal axis shows the distance from the substrate surface (height). That is, in this simulation, the thickness of the electrode is set to 5 μm, so that the portion below 5 μm on the horizontal axis shows the region sandwiched between the working electrode and the reference electrode, and the portion above 5 μm shows the region above it. .

In FIG. 8, the electric field _Er in the same diameter direction has a large value, and has a maximum near the working electrode. This suggests that a strong horizontal electric field is generated between the electrodes when there is a corner portion in the electrodes. Therefore, this suggests that, for example, when biopolymers such as nucleic acids are subjected to dielectrophoresis, the biopolymers are concentrated on the corners of the working electrode due to a strong horizontal electric field.

  On the other hand, for example, when the electrode is embedded in the substrate and the working electrode is formed in an edge-free manner, it can be assumed that the horizontal electric field hardly acts on a biopolymer such as a nucleic acid. Therefore, since the concentration of the electric field at the corner portion can be reduced, it can be estimated that a highly uniform electric field can be formed.

FIG. 9 shows the simulation result of the potential distribution in the vertical direction.
In the figure, the horizontal axis (“Position”) indicates the height (unit: μm) with respect to the substrate surface, and the vertical axis (“Potential”) indicates the potential (unit: V).
In the figure, the plot of “Conventional electrodes” indicates the potential distribution when the electrode having a corner portion is simulated, and the plot of “Embedded electrodes” indicates the potential distribution when the edge-free electrode is simulated.

In FIG. 9, in the region sandwiched between the electrodes (height 0 to 5 μm), the potential is equal to the applied voltage 2 V, and in the upper electrolyte solution (height 5 μm or more), the potential gradually decreases. Yes. In addition, in the case of an electrode having a corner portion and the case of an edge-free electrode, the potential distribution is almost equal in a region (height of 5 μm or more) away from the region sandwiched between both electrodes.
This suggests that even when the electrodes are formed in an edge-free manner, a potential distribution almost similar to that in the case of using the electrodes having corner portions is generated.
Therefore, this simulation result suggests that when the electrode is formed in an edge-free manner, an electric field can be formed as in the conventional case, and the concentration of the electric field on the corner portion can be suppressed.
In the simulation result of FIG. 9, since the potential is approximately 0 V at a point of about 15 μm from the working electrode, the vertical electric field is 2 V / 15 μm = 1.3 × 10 ⁶ V / m. This value is sufficient for performing dielectrophoresis.

FIG. 10 shows a simulation result of the potential distribution in the vertical direction when three values of the electrode thickness d, the gap g between the electrodes, and the width w of the reference electrode are varied.
In the figure, the horizontal axis (“Position”) indicates the height (unit: μm) with respect to the substrate surface, and the vertical axis (“Potential”) indicates the potential (unit: V).

This simulation was performed assuming values shown in the following Table 1 as parameters.

As a result, in FIG. 10, as in FIG. 9, in the region sandwiched between the electrodes (height 0 to 0.1 μm or 0 to 1 μm), the potential is equal to the applied voltage 2 V, and the electrolyte solution above it In the middle (the height is 0.1 μm or 1 μm or more), the potential gradually decreases. In any simulation result, the potential distribution is almost equal in the region (height of 0.1 μm or 1 μm or more) away from the region sandwiched between both electrodes.
This suggests that even when each structural parameter is appropriately changed, an electric field can be formed as in the conventional case, and concentration of the electric field on the corner portion can be suppressed. Therefore, this experimental result suggests that the degree of freedom in electrode design can be increased by forming the electrode in an edge-free manner.
In the simulation result of FIG. 10, since the electric potential becomes approximately 0 V at a point of about 15 μm from the working electrode, the electric field in the vertical direction is 2 V / 15 μm = 1.3 × 10 ⁶ V / m. This value is sufficient for performing dielectrophoresis.

  The present invention can be applied to a bioassay substrate such as a DNA chip, and is useful in both the manufacturing stage of the bioassay substrate (stage of immobilizing a detection substance) and the interaction detection / measurement stage.

The cross-sectional schematic diagram which shows the example of the interaction detection part A which concerns on this invention. The cross-sectional schematic diagram which shows another example of the interaction detection part A which concerns on this invention. The cross-sectional schematic diagram which shows another example of the interaction detection part A which concerns on this invention. The cross section and upper view schematic diagram which show another example of the interaction detection part A which concerns on this invention. The figure which shows the electrode structure used for simulation. The figure shown about the parameter used for simulation. The figure which shows the simulation result of electric field distribution. The figure which shows the simulation result which divided and divided the electric field distribution in the case where a corner | angular part exists in an electrode into the same-diameter direction and a perpendicular direction. The figure which shows the simulation result of the electric potential distribution of a perpendicular direction. The figure which shows the simulation result of the electric potential distribution of the orthogonal | vertical direction at the time of shaking three values, the thickness d of an electrode, the gap g between electrodes, and the width w of a reference electrode.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 11 Substrate surface 2 Reaction field 3 Detection substance 4 Wiring A Interaction detection part F Electric field R Reference electrode W Working electrode

Claims (5)

A working electrode formed in an edge-free region in the central region of the substrate surface facing the reaction field of the interaction between the detection substance and the target substance, and formed in a substantially ring shape at a position surrounding the working electrode, An interaction detecting unit that provides a reference electrode arranged in parallel with a free edge on the same substrate surface as the working electrode .   The interaction detection unit according to claim 1, wherein the detection substance is fixed to the working electrode. The discontinuous portion provided in a part of the substantially ring-shaped, the partial interaction detector according to claim 1 or 2, characterized in that extending the wiring connected to the working electrode. A working electrode formed in an edge-free region in the central region of the substrate surface facing the reaction field of the interaction between the detection substance and the target substance, and formed in a substantially ring shape at a position surrounding the working electrode, A bioassay substrate comprising an interaction detector provided with a reference electrode arranged in parallel with a free edge on the same substrate surface as the working electrode . In the following procedure (1) or (2),
A working electrode formed in an edge-free region in the central region of the substrate surface facing the reaction field of the interaction between the detection substance and the target substance, and formed in a substantially ring shape at a position surrounding the working electrode, A highly uniform electric field is formed in the reaction field by using the working electrode and the reference electrode in the interaction detection unit provided with the reference electrode arranged in parallel with the free edge on the same substrate surface as the working electrode, and the detection substance or A method of dielectrophoresis of a target substance toward a working electrode.
(1) Procedure for immobilizing the detection substance on the working electrode.
(2) A procedure for causing the detection substance fixed on the working electrode to interact with the target substance.