JP2007315955A - Detection element, manufacturing method of the same, and target detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection element for accurately detecting various targets, such as proteins, its manufacturing method, and a target detector that uses the detection element. <P>SOLUTION: The target detector 10 comprises the detection element 20, an excitation light source 11, a light-detecting section 12, a data processing section 13, and a power supply 16. The detection element 20 comprises a substrate 21, a cover 22 attached to the substrate 21, a flow path 22a formed on a face of the cover 22 on the substrate 21 side and has a sample solution 29 flowing, and an Au electrode 25 and a Pt electrode 26 disposed on a surface of the substrate 21 and exposed to the flow path 22a. A target detecting body 30 for acquiring a target is bonded to a surface of the Au electrode 25. The Au electrode 25 is fixed to the substrate 21 via an electrode adhesive layer 24, comprising a material having molecules containing a thiol group. The substrate 21 and the cover 22 are attached via a substrate adhesion layer 23 that comprises molecules containing a silanol group and an amino group. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a detection element capable of detecting various targets such as proteins with high sensitivity, a manufacturing method thereof, and a target detection apparatus using the detection element.

  2. Description of the Related Art In recent years, so-called genome drug discovery has been promoted in which drug development is efficiently performed using genome information related to nucleotide sequences, gene expression information, proteins, and the like obtained by rapid progress in human genome analysis research. In genomic drug discovery, genes related to diseases are searched based on genomic information, and the mechanism of pathogenesis is clarified from the analysis, and target molecules such as proteins most effective for treatment are determined. In addition, a drug candidate compound having a medicinal effect on the target molecule is designed and synthesized. In such a process, the importance of an apparatus capable of easily detecting a target molecule such as a protein is increasing.

On the other hand, the Au thin film emits evanescent light in the visible light region, and self-organization occurs in which various molecules spontaneously form high-density and highly-oriented molecular films on the surface. The formation of functionalized monolayers (SELF-Assembled Monolayers: SAMs) attracts attention as a material capable of constructing the function of a protein detection sensor (see, for example, Patent Document 1). The Au thin film is formed on the surface of the glass substrate, and a detection unit in which an antibody or the like for capturing a target molecule is bound to the molecular chain of the polynucleotide is self-organized on the surface of the Au thin film. The protein detection sensor can detect the molecular weight distribution and density of the target molecule optically or electrochemically by immersing the Au thin film to which the detection unit is bound in a sample solution containing the target molecule and emitting light from the detection unit. .
JP 2003-202337 A

  By the way, in order to increase the efficiency of protein detection, it is desired to increase the sensitivity of the protein detection sensor. That is, it is desired to detect the target molecule of interest with a small amount of sample solution. Therefore, it is necessary to arrange the Au thin film and the detection unit in a minute space with high density. Since the Au thin film has poor adhesion to the glass substrate, a Cr film or a Ti film is used as the adhesive layer. However, in order to form a minute space with a glass substrate, it is necessary to bond the glass substrates together. For this reason, thermal fusion with a heating temperature of 1000 ° C. or higher is used. When exposed to such a high temperature, the Cr film and the Ti film diffuse into the Au thin film, the electrical conductivity of the Au film decreases, and further, the problem that the SAMs of the detection unit cannot be stably formed occurs. Further, a strong acid on the surface of the glass substrate necessary for the formation of stable SAMs without using an adhesive film such as a Cr film or a Ti film, such as a so-called piranha solution (sulfuric acid: hydrogen peroxide solution = 3: 1), 60 When the pretreatment with% nitric acid is used, there is a problem that if the ultrasonic thin film is used after the Au thin film is formed, the Au thin film is easily peeled off from the glass substrate, resulting in poor reliability.

  Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a detection element capable of detecting various targets such as proteins with high sensitivity, a manufacturing method thereof, and a target detection apparatus using the detection element. Is to provide.

According to one aspect of the present invention, a first substrate, a second substrate bonded to the first substrate via a first adhesive layer, and the first substrate and the second substrate. Any one of the channels formed on the surfaces bonded to each other, the first electrode and the second electrode provided in the channel,
A target detector coupled to the first electrode and capable of capturing a target in the sample solution flowing through the flow path, wherein the first adhesive layer is made of a material containing molecules having a silanol group and an amino group. The first electrode is fixed to the surface of the first substrate via a second adhesive layer, and the second adhesive layer is a detection element made of a material containing a molecule having a silanol group and a thiol group. Provided.

  According to this invention, it has the structure where the 1st board | substrate and the 2nd board | substrate were bonded together, and the flow path is formed in the structure. Further, an Au electrode to which a target detector for capturing the target is bound is provided in the flow path. Since the Au electrodes can be arranged in the flow path with high density, the detection element can detect the target with high sensitivity. Further, the detection element can be extremely miniaturized.

  According to another aspect of the present invention, a step of selectively forming a first electrode on a process substrate and an adhesive layer including molecules having a silanol group and a thiol group are formed on the surface of the first electrode. A step of bonding the first substrate to the first electrode through an adhesive layer, a step of removing the step substrate from the first electrode, and a step of the first electrode side of the first substrate. Bonding the step of forming another adhesion layer containing molecules having silanol groups and amino groups on the surface, the first substrate and the second substrate on which the flow path is formed, via another adhesion layer And a step of forming a second electrode in the flow path, and a step of binding a target detector capable of capturing a target to the first electrode. .

  According to the present invention, since the first substrate and the second substrate are bonded by another adhesive layer containing a molecule having a silanol group and an amino group, the first substrate and the second substrate can be bonded at a lower temperature than in the past and already formed. This does not adversely affect the Au electrode.

  According to another aspect of the present invention, the detection element according to claim 1, a voltage applying unit that applies a voltage between the first electrode and the second electrode, and a first of the detection element. There is provided a target detection apparatus comprising: a light irradiation means for irradiating light toward the electrode; and a light detection means for detecting light emitted from the target detection body upon receiving the light emitted from the light irradiation means. The

  According to the present invention, a target detection apparatus with high target detection sensitivity can be realized. Further, the target detection device can be reduced in size and integrated.

  ADVANTAGE OF THE INVENTION According to this invention, the detection element which can detect various targets, such as protein, with high sensitivity, its manufacturing method, and the target detection apparatus using this detection element can be provided.

  Embodiments will be described below with reference to the drawings.

  FIG. 1 is a schematic configuration diagram of a detection device according to an embodiment of the present invention, and FIG. 2 is an exploded perspective view of a detection element according to an embodiment of the present invention that constitutes the detection device.

  1 and 2, the target detection apparatus 10 includes a detection element 20, an excitation light source 11, a light detection unit 12, a data processing unit 13, a power source 16, and the like.

  The detection element 20 is formed on the substrate 21, the cover 22 bonded to the substrate 21, the surface of the cover 22 on the substrate 21 side, the flow path 22 a through which the sample solution 29 circulates, and the surface of the substrate 21. The Au electrode 25 and the Pt electrode 26 exposed to the flow path 22a, and the pad electrodes 28a and 28b connected to the Au electrode 25 and the Pt electrode 26 by the wiring patterns 27a and 27b, respectively. Further, a target detector 30 that captures the target is coupled to the Au electrode 25. The Au electrode 25 is fixed to the substrate 21 through the electrode adhesive layer 24. Furthermore, the substrate 21 and the cover 22 are bonded via a substrate adhesive layer 23. Further, a reference electrode 38 is provided so as to be exposed to the flow path, and the reference electrode 38 is electrically connected to the Au electrode 25 via a voltmeter 39. Note that feedback for reducing or reducing the voltage difference from the voltmeter 39 to the power source 16 may be applied based on the voltage difference between the voltage measured by the voltmeter 39 and a preset voltage.

  As the substrate 21, for example, a glass substrate (for example, a tempered glass substrate or a crystallized glass substrate), a quartz glass substrate, a silicon substrate, or the like can be used. When the excitation light is applied to the Au electrode 25 through the cover 22, the substrate 21 may be an opaque or translucent substrate. Moreover, when using a silane coupling agent as a material of the board | substrate adhesion layer 23 mentioned later, it is preferable to use a glass substrate and quartz glass at the point which can be bonded firmly.

  The cover 22 is made of a transparent substrate that transmits excitation light, and a glass substrate (for example, a tempered glass substrate or a crystallized glass substrate), a quartz glass substrate, or the like can be used.

  The cover 22 has a groove formed on the surface on the substrate 21 side. As for the groove part, the flow path 22a is formed when the cover 22 and the board | substrate 21 are bonded. In FIG. 1, the cross-sectional shape of the groove is indicated by a rectangle, but is not particularly limited. A sample solution supply port 22b and a sample solution discharge port 22c communicating with the groove are formed on the surface of the cover 22. The sample solution 29 is supplied from the sample solution supply port 22b by a pump or the like, passes through the flow path 22a, contacts the target detection body 30, the Au electrode 25, and the Pt electrode 26, and is discharged from the sample solution discharge port 22c. .

  A substrate adhesive layer 23 is provided between the substrate 21 and the cover 22. For the substrate adhesive layer 23, it is preferable to use a material containing a molecule having an amino group and a silanol group, and it is preferable to use a silane coupling agent having an amino group. Thereby, the board | substrate 21 and the cover 22 are adhere | attached firmly, and it becomes possible to adhere | attach at the heating temperature extremely lower than before in the case of adhesion | attachment so that it may demonstrate later.

  FIG. 3 is an explanatory view showing an example of a substrate adhesive layer. Referring to FIG. 3, in the substrate adhesive layer 23, silanol groups are bonded to the substrate 21, amino groups are bonded firmly to the cover 22, and the substrate 21 and the cover 22 are bonded firmly. Examples of the silane coupling agent suitable for the substrate adhesive layer 23 include materials having the following general formula.

NH ₂ (CH ₂ ) _x [(CH ₂ ) _l (CHR) _m (CH ₂ ) _n ] _y (CH ₂ ) _z SiOH (1)
In the above formula (1), x, y, 1, m, and n are each an integer of 0 to 18, and z is an integer of 1 to 18. R is H or a phenyl group. For example, when m is 0 and x and y are 1 or more, the molecule represented by the above formula (1) has a structure in which a linear alkyl group extends in one direction. In this case, since the molecules tend to be bundled, silanol groups and amino groups are localized. Therefore, the silanol group is firmly bonded to the substrate 21. On the other hand, the amino group is strongly bonded by the electrostatic force between the polarization δ ⁺ and the polarization δ ⁻ of the OH group on the surface of the cover 22.

  For example, when m is 1 and R is a phenyl group, the phenyl groups of each molecule exert a hydrophobic interaction with each other and the attractive force of the intermolecular force with each other to form a bundle of molecules. It becomes easy and the effect similar to the above is acquired.

  The target detection body 30 includes an interaction unit 31, a light emitting unit 32, and a coupling unit 33, and further includes other units (not shown) appropriately selected as necessary.

  The interaction unit 31 is not particularly limited as long as it includes a region capable of electrical interaction with the Au electrode 25 and one end includes a region capable of being coupled to the Au electrode 25, and can be appropriately selected depending on the purpose. A specific example of the interaction part 31 is an ionic polymer in that an electrical interaction is possible. The ionic polymer is preferably selected from positively charged ionic polymers (positive ionic polymers) and negatively charged ionic polymers (negative ionic polymers). Examples of the positive ion polymer include peptide nucleic acid (PNA), guanidine DNA, polyamine, and the like. Examples of the negative ion polymer include polynucleotide, polyphosphate, and the like. These negative ionic polymers are preferable in terms of easy control of electrical interaction with the Au electrode 25 in that negative charges are present in the molecule at regular intervals. As the polynucleotide, DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) can be appropriately selected. In FIG. 1 and FIGS. 5 and 6 described later, double-stranded DNA is shown as an example, and the interaction part 31 is negatively charged. In addition, there is no restriction | limiting in particular as a method of producing polynucleotide, What is necessary is just to select suitably from a well-known method (for example, method using a DNA automatic synthesizer).

The interaction part 31 has a site | part couple | bonded with the Au electrode 25 in the end. A suitable example of this site is a thiol group (—S—). The thiol group is firmly bonded to the Au electrode 25 (or an Al electrode, a TiO ₂ electrode, or an Ag electrode as an alternative to the Au electrode 25). For this reason, when the pulse voltage which alternates to Au electrode 25 is applied, even when target detection body 30 repeats adsorption | suction and repulsion with Au electrode 25, it is preferable at the point that a coupling | bonding is hard to cut | disconnect. Moreover, it is not limited to a thiol group, You may select another group suitably.

  The light emitting unit 32 is coupled to a predetermined position, for example, one end of the interaction unit 31. The light emitting part 32 emits light when the excitation light is irradiated and the interaction part 31 does not interact with the Au electrode 25, and the interaction part 31 interacts with the Au electrode 25. When light is emitted, there is no particular limitation as long as light emission is weakened or light is not emitted, and can be appropriately selected according to the purpose. Examples of the light emitting unit 32 include fluorescent dyes and metals. Here, as an example, the light emitting section 32 will be described as cyanine-based Cy3 (trademark) as a fluorescent dye.

  While the fluorescent dye is interacting close to the Au electrode 25, the light emission is weakened or does not emit light even when irradiated with light having an absorbable wavelength. The fluorescent dye can emit light by excitation light when it is separated from the Au electrode 25 and does not interact.

  The binding portion 33 is not particularly limited as long as the target can be captured, and can be appropriately selected according to the purpose. There is no restriction | limiting in particular as a capture | acquisition aspect, Adsorption, a chemical bond etc. are mentioned. Examples of the binding portion 33 include an antibody against a target, an antigen, an enzyme, a coenzyme, and the like. Moreover, there is no restriction | limiting in particular as a target, According to the objective, it can select suitably. Examples of the target include organic molecules such as proteins, lipoproteins, glycoproteins, polypeptides, lipids, polysaccharides, lipopolysaccharides, nucleic acids, and drugs.

  Examples of the sample solution 29 containing the target include pathogens such as bacteria and viruses, cell fluids separated from living bodies, lymph fluid, blood, and the like, and cell destruction after concentration directly or as necessary. The thing which gave the process previously can be used.

The Au electrode 25 is formed on the substrate 21 via the electrode adhesive layer 24, and has a thickness of, for example, 100 nm. The surface of the Au electrode 25 is exposed to the flow path 22a. The Au electrode 25 has a thickness of, for example, 100 nm, is combined with the target detection body 30 described above, and functions as one electrode that applies an electric field to the target detection body 30. Instead, a conductive material can be used for the Au electrode 25. For example, an Al electrode, a TiO ₂ electrode, or an Ag electrode may be used.

  The electrode adhesive layer 24 adheres the surface of the substrate 21 and the Au electrode 25. As the electrode adhesive layer 24, it is preferable to use a material including a molecule having a silanol group and a thiol group. The silanol group of the electrode adhesive layer 24 is bonded to the substrate 21, and Au is firmly bonded to the thiol group. As the electrode adhesive layer 24, for example, a silane coupling agent having a thiol group can be used.

  FIG. 4 is an explanatory view showing an example of an electrode adhesive layer. Referring to FIG. 4, when the substrate 21 is a glass substrate or a quartz substrate, the electrode adhesive layer 24 has an extremely strong silanol group bonded to the substrate 21, so that the substrate 21 and the Au electrode 25 are firmly fixed. Is done. Here, the silanol group and the thiol group are connected by a straight chain, but are not limited thereto. For example, a silane coupling agent suitable for the electrode adhesive layer 24 includes a material having the following general formula.

HS (CH ₂ ) _x [(CH ₂ ) _l (CHR) _m (CH ₂ ) _n ] _y (CH ₂ ) _z SiOH (2)
In the above formula (2), x, y, 1, m, and n are each an integer of 0 to 18, and z is an integer of 1 to 18. R is H or a phenyl group. For example, when m is 0 and x and y are 1 or more, the molecule represented by the above formula (2) has a structure in which a linear alkyl group extends in one direction. In this case, since the molecules tend to be bundled, silanol groups and thiol groups are localized. Therefore, the silanol group is firmly bonded to the substrate 21. On the other hand, the thiol group is firmly bonded to the Au electrode 25.

  For example, when m is 1 and R is a phenyl group, the phenyl groups of each molecule exert a hydrophobic interaction with each other and the attractive force of the intermolecular force with each other to form a bundle of molecules. It becomes easy and the effect similar to the above is acquired.

  Returning to FIG. 1 and FIG. 2, the Au electrode 25 is drawn out to the electrode pad 28a through the wiring pattern 27a, but the wiring pattern 27a and the pad electrode 28a may also be formed of Au.

  The Pt electrode 26 is directly formed on the substrate 21 and has a thickness of, for example, 80 nm. The surface of the Pt electrode 26 is exposed to the flow path 22 a and is formed at a predetermined distance from the Au electrode 25. The Pt electrode 26 functions as one electrode that applies an electric field to the target detection body 30 as a counter electrode. Further, since the Pt electrode 26 has good adhesion to the substrate 21, it is not necessary to form the electrode adhesive layer 24.

  The reference electrode 38 becomes a reference potential when the voltage applied to the Au electrode 25 is measured by the voltmeter 39. Examples of the reference electrode 38 include an Ag / AgCl / 3M KCl and a saturated carmello electrode.

  The excitation light source 11 is not particularly limited as long as it can irradiate light toward the Au electrode 25 and can be appropriately selected according to the purpose. The excitation light source 11 is preferably a light source that emits visible light, for example, a xenon lamp, a laser, or the like. Furthermore, an optical system for selectively irradiating each Au electrode 25 with excitation light, such as an optical fiber, may be used. The light emitted from the excitation light source 11 passes through the cover 22 made of a transparent material, and is irradiated to the Au electrode 25 and the target detection body 30.

  The light detection unit 12 is not particularly limited as long as it can detect light emitted from the target detection body 30 that has received light emitted from the excitation light source 11, and can be appropriately selected according to the purpose. For example, a light receiving sensor, a CCD camera, a photomultiplier, or a photodiode (for example, an avalanche photodiode (APD)) can be used as the light detection unit 12.

  The data processing unit 13 analyzes the weight of the target captured by the coupling unit 33 based on the data of the light intensity change from the light detection unit 12 and the time change of the applied voltage, and the result is input to the input / output unit 14. The data is output to a monitor or a printer or recorded in the storage device 15.

  As the power source 16, for example, a pulse power source is used. The power supply 16 applies a pulse voltage in which positive and negative voltages alternate between the Au electrode 25 and the Pt electrode 26 via the pad electrodes 28a and 28b and the wiring pattern shown in FIG. Apply.

  Next, the operation of the target detection apparatus 10 will be described.

5A and 5B are explanatory diagrams for detecting a target. FIG. 5A shows a case where a negative voltage is applied to the Au electrode 25 with respect to the Pt electrode 26, and FIG. 5B shows a case where a positive voltage is applied. 6 is a relationship diagram between the applied voltage and the fluorescence intensity, and the vertical axis of FIG. 6 (A) indicates the applied voltage measured by the voltmeter shown in FIGS. 5 (A) and 5 (B). The applied voltage indicates the voltage of the Au electrode 25 with respect to the reference electrode 38. The applied voltage is shown shifted by the balanced electrode potential V _E of the reference electrode, but substantially the same voltage is applied to each of positive and negative. In addition, the vertical axis in FIG. 6B indicates the fluorescence intensity.

  5 and 6 together with FIG. 1, the pulse voltage shown in FIG. 6A is applied between the Pt electrode 26 and the Au electrode 25 while irradiating the Au electrode 25 with excitation light. As shown in FIG. 5A, when a negative voltage is applied to the Au electrode 25, the interaction unit 31 is negatively charged, and thus is substantially upright so as to be separated from the Au electrode 25. At this time, since the light emitting portion 32 is separated from the Au electrode 25, the light emitting portion 32 emits light by the energy of excitation light.

  Next, as illustrated in FIG. 5B, when a positive voltage is applied to the Au electrode 25, the interaction unit 31 is negatively charged and thus is close to the Au electrode 25. At this time, since the light emitting portion 32 is close to the Au electrode 25, the light emission is weakened or does not emit light.

As described above, by applying the pulse voltage alternately, the change in the fluorescence intensity shown in FIG. The fluorescence intensity decreases from the strongest fluorescence intensity I ₁ (fluorescence intensity in the state of FIG. 5A) and changes to the weakest fluorescence intensity I ₂ (fluorescence intensity in the state of FIG. 5B). The change in fluorescence intensity at this time depends on the molecular weight of the captured target. That is, the larger the target molecular weight, the slower the rate of change. The change in the fluorescence intensity is analyzed by the data processing unit 13, and an analysis result such as the molecular weight of the captured target and the number of captured targets is obtained. As described above, the target detection apparatus 10 can detect and analyze the target in the sample solution 29.

  The detection element 20 according to the present embodiment has a structure in which a substrate 21 and a cover 22 are bonded, and a fine flow path 22a (for example, a width and a height of 100 μm) is formed in the structure. Further, an Au electrode 25 (for example, 50 μm on one side) to which a target detection body 30 that captures the target is coupled is provided in the flow path 22a. Since the Au electrodes 25 can be arranged at high density in the flow path 22a, the detection element 20 can detect the target with high sensitivity. Further, the detection element 20 can be extremely miniaturized.

  Further, since the Au electrode 25 is firmly bonded to the substrate 21 by the electrode adhesive layer 24, the Au electrode 25 is hardly peeled off from the substrate 21. Therefore, the detection element 20 is excellent in reliability and durability. Therefore, since the detection element 20 can be used repeatedly, cost is reduced.

  In the above description, the Au electrode 25 is formed on the surface of the substrate 21 and the groove portion that becomes the flow path 22a is formed on the cover 22. However, the groove portion that becomes the flow path 22a is formed on the surface of the substrate 21, and the bottom of the groove portion is formed. The Au electrode 25 may be formed.

  Moreover, the target detection apparatus 10 according to the present embodiment can realize an apparatus with high target detection sensitivity. Further, the target detection device 10 can be downsized.

  Next, a method for manufacturing the detection element according to the embodiment will be described with reference to FIGS. 7 and 8 are manufacturing process diagrams of the detection element. For convenience of explanation, FIGS. 8A to 8C are shown by replacing the top and bottom with respect to FIGS. 7A to 7D.

  7A, a sacrificial film 42 is formed on the process substrate 41 by a spin coating method, a spray method, a vacuum deposition method, a sputtering method, a CVD method, or the like. The sacrificial film 42 is not particularly limited as long as it is a material that has etching selectivity with the Au electrode 25 and does not dissolve in each other. For example, a methylsiloxane film or a silicon oxide film can be used. The thickness of the insulating film is set to be equal to or thinner than the Au electrode 25 formed in the next step.

  In the step of FIG. 7A, a resist film 43 is further formed on the sacrificial film. Next, the resist film 43 is patterned by photolithography to form an opening at a position where the Au electrode 25 is to be formed. An opening for forming a wiring pattern and a pad electrode connected to the Au electrode 25 shown in FIG. 2 is also formed. Since the wiring pattern and the pad electrode are formed in the same manner as the Au electrode 25 in the next step, the Au electrode 25 includes the wiring pattern and the pad electrode unless otherwise specified. Further, the sacrificial film 42 is etched using the resist film 43 in which the opening is formed as a mask to form the opening. This etching is not particularly limited as long as the sacrificial film 42 can be etched. For example, a wet etching method is used.

  7B, an Au film having a thickness of, for example, 100 nm is formed on the surface of the structure shown in FIG. 7A by a vacuum evaporation method, a sputtering method, or the like. Further, the excess Au film formed on the resist film 43 is lifted off together with the resist film 43. In this way, the Au electrode 25 selectively formed on the sacrificial film 42 is formed.

  Next, in the step of FIG. 7C, the electrode adhesive layer 24 is formed on the surface of the sacrificial film 42 and the Au electrode 25. Specifically, a material containing a molecule having a silanol group and a thiol group on the surface of the sacrificial film 42 and the Au electrode 25, for example, a silane coupling agent having a thiol group is diluted in an organic solvent, spin coating method, spraying method, etc. Apply by. Further drying is performed to remove the organic solvent.

  In the step of FIG. 7C, the substrate 21 is further aligned with the process substrate 41. Further, the substrate is pressed against the process substrate 41 through the electrode adhesive layer 24 to bond the substrate 21 and the process substrate 41 together. At this time, heating is not necessary. The silanol group of the electrode adhesive layer 24 and the substrate 21 are strongly bonded. Note that a Pt electrode 26 and a wiring pattern and electrode pads (shown in FIG. 2) connected to the Pt electrode 26 may be formed in advance on the surface of the substrate 21. Since the Pt electrode 26 has good adhesion to the substrate 21, particularly a glass substrate, it can be formed, for example, by sputtering.

  7D, the sacrificial film 42 is dissolved by wet etching, and the process substrate 41 is peeled from the Au electrode 25, the electrode adhesive layer 24, and the substrate 21 structure. As the wet etching solution, for example, when the sacrificial film 42 is a methylsiloxane film or silicon oxide, hydrofluoric acid can be used.

  Next, in the step of FIG. 8A, the portion other than the portion to which the Au electrode 25 is bonded is removed from the electrode bonding layer 24 of the structure of FIG. 7D by ashing.

  In the step of FIG. 8A, a substrate adhesive layer 23 is further formed on the surface of the substrate 21. Specifically, a material containing a molecule having a silanol group and an amino group, for example, a silane coupling agent having an amino group is diluted in an organic solvent and applied to the surface of the substrate 21 by a spin coating method, a spray method, or the like. Further drying is performed to remove the organic solvent.

  Next, in the step of FIG. 8B, the cover 22 is positioned with respect to the structure of FIG. A groove 22a, a sample solution supply port 22b, and a sample solution discharge port 22c are formed in advance in the cover 22 as a flow path shown in FIG. After positioning the cover 22, the cover 22 is bonded to the substrate 21. In this bonding, the cover 22 is pressed against the substrate 21 through the substrate adhesive layer 23 and is heated to a temperature in the range of 150 ° C. to 450 ° C. (preferably 150 ° C. to 250 ° C.). Thereby, the substrate 21 and the cover 22 are firmly bonded via the substrate bonding layer 23. In the substrate adhesive layer 23, an amino group causes a dehydration reaction with an OH group on the surface of the cover 22, and, for example, when the cover 22 is a glass substrate, it bonds to silicon atoms of the glass substrate. As a result, the Au electrode 25 and the Pt electrode 26 are disposed in the flow path 22a, and the flow path 22a is formed.

  Next, in the step of FIG. 8C, a buffer solution (for example, Tris-HCl (pH 7.5)) is flowed into the flow path 22a, and the target detection body 30 synthesized in advance is mixed with the buffer solution to flow. Pour into channel 22a and let stand for about 180 minutes. As a result, the thiol group of the target detector 30 is firmly bonded to the surface of the Au electrode 25. Further, a buffer solution is injected into the flow path 22a to remove the excess target detection body 30 in the flow path 22a. Thus, a detection element is formed.

  In this manufacturing method, since the substrate 21 and the cover 22 are bonded by the substrate adhesive layer 23, the Au electrode 25 already formed is not adversely affected. In particular, by using a silane coupling agent having an amino group for the substrate adhesive layer 23, the substrate 21 and the cover 22 can be bonded in a lower temperature range (150 ° C. to 450 ° C.) than before, and the Au electrode 25 For example, the target detector 30 is difficult to bind to the surface of the Au electrode 25, or the binding becomes unstable, which makes it difficult to form an aggregate of the target detector 30. Can be avoided.

  The Pt electrode 26 and its wiring pattern and pad electrode may be formed on the surface of the substrate 21 by vacuum deposition, sputtering, or CVD after removing the electrode adhesive layer 24 in the step of FIG. In this case, for example, it is selectively formed using a resist film patterned using a photolithography method or a metal mask.

  The preferred embodiment of the present invention has been described in detail above, but the present invention is not limited to the specific embodiment, and various modifications can be made within the scope of the present invention described in the claims.・ Change is possible.

In addition, the following additional notes are disclosed regarding the above description.
(Appendix 1) a first substrate;
A second substrate bonded to the first substrate via a first adhesive layer;
A flow path formed on the surface of one of the first substrate and the second substrate bonded to each other;
A first electrode and a second electrode provided in the flow path;
A target detector coupled to the first electrode and capable of capturing a target in the sample solution flowing through the flow path,
The first adhesive layer is made of a material containing a molecule having a silanol group and an amino group,
The detection element, wherein the first electrode is fixed to the surface of the first substrate via a second adhesive layer, and the second adhesive layer is made of a material containing a molecule having a silanol group and a thiol group.
(Additional remark 2) The said 1st contact bonding layer consists of material containing the molecule | numerator which has a silanol group and an amino group as a terminal group, The detection element of Additional remark 1 characterized by the above-mentioned.
(Additional remark 3) The said 1st contact bonding layer is shown by following General formula (1), The detection element of Additional remark 1 characterized by the above-mentioned.

NH ₂ (CH ₂ ) _x [(CH ₂ ) _l (CHR) _m (CH ₂ ) _n ] _y (CH ₂ ) _z SiOH (1)
(However, x, y, l, m, and n are each an integer of 0 to 18, and z is an integer of 1 to 18. R is H or a phenyl group.)
(Additional remark 4) The said 2nd contact bonding layer consists of material containing the molecule | numerator which has a silanol group and a thiol group as a terminal group, The detection element of Additional remark 1 characterized by the above-mentioned.
(Additional remark 5) The said 2nd contact bonding layer is shown by following General formula (2), The detection element of Additional remark 1 characterized by the above-mentioned.

HS (CH ₂ ) _x [(CH ₂ ) _l (CHR) _m (CH ₂ ) _n ] _y (CH ₂ ) _z SiOH (2)
(However, x, y, l, m, and n are each an integer of 0 to 18, and z is an integer of 1 to 18. R is H or a phenyl group.)
(Supplementary note 6) The detection element according to supplementary note 1, wherein one of the first substrate and the second substrate is made of a transparent substrate material.
(Appendix 7) The second substrate is made of a transparent substrate material, and the flow path is defined on a groove formed in the second substrate and a surface of the first substrate,
The detecting element according to claim 1, wherein the second electrode is formed on the surface of the substrate at a distance from the first electrode.
(Additional remark 8) The said target detection body can be light-emitted when irradiated with light, when the interaction part which can interact with a 1st electrode, and this interaction part is not interacting with a 1st electrode The detection element according to appendix 1, wherein the detection element includes a light emitting unit and a detection unit capable of capturing a target.
(Additional remark 9) The said interaction part is an ionic polymer, The detection element of Additional remark 8 characterized by the above-mentioned.
(Additional remark 10) The said light emission part is a fluorescent dye, The detection element of Additional remark 8 characterized by the above-mentioned.
(Appendix 11) A step of selectively forming a first electrode on a process substrate;
Forming an adhesive layer including a molecule having a silanol group and a thiol group on the surface of the first electrode;
Bonding the first substrate to the first substrate through an adhesive layer;
Removing a process substrate from the first electrode;
Forming another adhesive layer containing molecules having a silanol group and an amino group on the surface of the first substrate on the first electrode side;
Bonding the first substrate and the second substrate on which the flow path is formed via another adhesive layer;
Forming a second electrode in the flow path;
And a step of binding a target detection body capable of capturing a target to the first electrode.
(Additional remark 12) The process of forming the said contact bonding layer apply | coats the silane coupling agent containing a thiol group to the surface of a 1st electrode, The manufacturing method of the detection element of Additional remark 11 characterized by the above-mentioned.
(Additional remark 13) The process of forming the said other contact bonding layer apply | coats the silane coupling agent containing an amino group to the surface by the side of the 1st electrode of a 1st board | substrate of the additional remark 11 characterized by the above-mentioned. Production method.
(Additional remark 14) The bonding process of said 1 board | substrate and a 2nd board | substrate sets to the range of 150 to 450 degreeC, and is bonded, The manufacturing method of the detection element of Additional remark 11 characterized by the above-mentioned.
(Supplementary Note 15) The step of selectively forming the first electrode on the process substrate includes:
A process of forming a sacrificial film on the process substrate;
A process of selectively forming openings in the surface of the sacrificial film;
Filling the opening with a first electrode material,
12. The method of manufacturing a detection element according to appendix 11, wherein the step of forming the adhesive layer covers the sacrificial film and the surface of the first electrode material.
(Supplementary Note 16) The detection element according to Supplementary Note 1,
Voltage applying means for applying a voltage between the first electrode and the second electrode;
Light irradiation means for irradiating light toward the first electrode of the detection element;
A light detection means for detecting light emitted from the target detection body in response to light emitted from the light irradiation means;

(Supplementary Note 17) The second substrate is made of a transparent substrate material,
The target detection apparatus according to appendix 16, wherein the light irradiation means or the light detection means irradiates or detects light through a second substrate.

  (Supplementary note 18) The target detection device according to supplementary note 17, wherein the second electrode is formed on the surface of the first substrate so as to be separated from the first electrode.

(Supplementary note 19) a first substrate;
A second substrate bonded to the first substrate via a first adhesive layer;
A flow path formed on a surface of one of the first substrate and the second substrate bonded to each other;
A first electrode and a second electrode provided in the flow path and capable of binding a target detector capable of capturing a target; and
The first adhesive layer is made of a material having molecules containing a silanol group and an amino group,
The first electrode is a structure that is fixed to the surface of the first substrate via a second adhesive layer, and the second adhesive layer is made of a material having molecules including a silanol group and a thiol group.

It is a schematic block diagram of the detection apparatus which concerns on embodiment of this invention. It is a disassembled perspective view of the detection element which concerns on embodiment of this invention. It is explanatory drawing which shows an example of a board | substrate adhesion layer. It is explanatory drawing which shows an example of an electrode contact bonding layer. (A) And (B) is explanatory drawing which detects a target. FIG. 5 is a relationship diagram between applied voltage and fluorescence intensity. (A)-(D) are manufacturing process drawings (the 1) of a detection element. (A)-(C) are manufacturing process drawings (the 2) of a detection element.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Target detection apparatus 11 Excitation light source 12 Optical detection part 13 Data processing part 14 Input / output part 15 Memory | storage device 16 Power supply 20 Detection element 21 Board | substrate 22 Cover 22a Flow path (groove part)
22b Sample solution supply port 22c Sample solution discharge port 23 Substrate adhesive layer 24 Electrode adhesive layer 25 Au electrode 26 Pt electrode 27a, 27b Wiring pattern 28a, 28b Pad electrode 29 Sample solution 30 Target detector 31 Interaction unit 32 Light emitting unit 33 Coupling Part 36 Target 41 Process substrate 42 Sacrificial film 43 Resist film

Claims (5)

A first substrate;
A second substrate bonded to the first substrate via a first adhesive layer;
A flow path formed on the surface of one of the first substrate and the second substrate bonded to each other;
A first electrode and a second electrode provided in the flow path;
A target detector coupled to the first electrode and capable of capturing a target in the sample solution flowing through the flow path,
The first adhesive layer is made of a material containing a molecule having a silanol group and an amino group,
The detection element, wherein the first electrode is fixed to the surface of the first substrate via a second adhesive layer, and the second adhesive layer is made of a material containing a molecule having a silanol group and a thiol group. The second substrate is made of a transparent substrate material, and the flow path is defined by a groove formed in the second substrate and a surface of the first substrate,
2. The detection element according to claim 1, wherein the second electrode is formed on the surface of the substrate at a distance from the first electrode. Selectively forming a first electrode on a process substrate;
Forming an adhesive layer including a molecule having a silanol group and a thiol group on the surface of the first electrode;
Bonding the first substrate to the first substrate through an adhesive layer;
Removing a process substrate from the first electrode;
Forming another adhesive layer containing molecules having a silanol group and an amino group on the surface of the first substrate on the first electrode side;
Bonding the first substrate and the second substrate on which the flow path is formed via another adhesive layer;
Forming a second electrode in the flow path;
And a step of binding a target detection body capable of capturing a target to the first electrode. The step of selectively forming the first electrode on the process substrate includes:
A process of forming a sacrificial film on the process substrate;
A process of selectively forming openings in the surface of the sacrificial film;
Filling the opening with a first electrode material,
The method for manufacturing a detection element according to claim 3, wherein the step of forming the adhesive layer is formed so as to cover surfaces of the sacrificial film and the first electrode material. The detection element according to claim 1 or 2,
Voltage applying means for applying a voltage between the first electrode and the second electrode;
Light irradiation means for irradiating light toward the first electrode of the detection element;
A light detection means for detecting light emitted from the target detection body in response to light emitted from the light irradiation means;

Images