JP2017044674A - Electrode, sensor, fluid device, and manufacturing method of electrode - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an electrode capable of reducing fluctuation of electric potential.SOLUTION: An electrode comprises: a first member having silver as a main component; a second member which has silver chloride as a main component and is in contact with at least a part of the first member; and a self-organized monomolecular film formed on the surface of the second member. The electrode may contain alkane thiol derivative in which the self-organized monomolecular film is chemically adsorbed on the surface of the second member. Further, a sensor comprises: a reference electrode having the first member having silver as a main component, the second member which has silver chloride as a main component and is in contact with at least a part of the first member, and the self-organized monomolecular film formed on the surface of the second member; and a working electrode including a detection part which detects biomolecules contained in solution being an object to be measured.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an electrode, a sensor, a fluidic device, and a method for manufacturing the electrode.

  In recent years, with the progress of miniaturization of chemical sensors and biosensors by semiconductor processing technology, development of minute electrodes capable of providing a stable potential has been required (for example, Patent Document 1 and Non-Patent Document 1). . For example, the reference electrode is an electrode that serves as a standard for potential measurement, and is an important component not only in basic research of electrochemistry but also in applied research such as biosensors. When detecting the target biomolecule or measuring the concentration by measuring the potential, the measurement is performed using the potential based on the reference electrode as an index. For this reason, in order to perform a stable and highly reliable measurement, it is necessary to use a reference electrode with a stable potential. According to the reference electrode described in Patent Document 1, a reference electrode that is entirely covered with a protective film and that part of the thin film skeleton metal pattern is exposed as an electrode terminal portion and is in contact with the solution to be measured is described. Has been.

JP 2001-4581 A

Hiroaki Suzuki and Takumi Taura, Thin-Film Ag / AgCl Structure and Operational Modes to Realize Long-Term Storage, Journal of The Electrochemical Society, 148 E468-E474 (2001).

  However, for example, the reference electrode described in Patent Document 1 has a problem that the potential of the reference electrode fluctuates due to dilution of the internal electrolyte due to the outflow of Cl ions.

  The present invention provides an electrode capable of reducing potential fluctuations.

One embodiment of the present invention provides the following (1) to (4).
(1) The electrode in one embodiment of the present invention includes a first member composed mainly of silver, a second member mainly composed of silver chloride and in contact with at least a part of the first member, A self-assembled monolayer formed on the surface of the second member.
(2) A sensor according to an embodiment of the present invention includes a first member mainly composed of silver, a second member mainly composed of silver chloride and in contact with at least a part of the first member, and the first member A reference electrode having a self-assembled monolayer formed on the surface of the member, and a working electrode having a detection unit for detecting an object contained in a solution to be measured.
(3) A fluid device according to an embodiment of the present invention includes the sensor described above.
(4) The method of manufacturing an electrode in one embodiment of the present invention includes a step of forming a layered first member made of silver or a silver alloy on one surface side of a substrate, and a surface of the first member. And a step of forming a layered second member made of silver chloride, and a step of forming a self-assembled monolayer on the surface of the second member.

It is a cross-sectional schematic diagram of the electrode of 1st Embodiment. It is a schematic diagram which shows the coupling | bonding of the AgCl layer and SAM film | membrane in the electrode of 1st Embodiment. 6 is a graph showing the result of XPS analysis of the surface of an AgCl layer before forming a SAM film in the electrode of the first embodiment. 6 is a graph showing the result of XPS analysis of the surface of an AgCl layer after forming a SAM film in the electrode of the first embodiment. In the electrode of 1st Embodiment, it is the SEM image which imaged the surface of the AgCl layer before forming a SAM film. 4 is an SEM image obtained by imaging the surface of the AgCl layer after forming the SAM film in the electrode of the first embodiment. It is a cross-sectional schematic diagram of the nucleic acid sensor of the second embodiment. It is a schematic diagram of the 1st capture probe employable as a capture probe fixed to the working electrode of 2nd Embodiment. It is a schematic diagram of the 2nd capture probe employable as a capture probe fixed to the working electrode of a 2nd embodiment. It is an exploded view of the fluid device of a 3rd embodiment. It is a schematic diagram of the fluid device of 4th Embodiment. It is a reaction schematic diagram of the analysis system in the sensing part (working electrode) of the biomolecule sensor of 5th Embodiment, and is a figure explaining the deposition reaction of silver. It is a reaction schematic diagram of the analysis system in the sensing part (working electrode) of the biomolecule sensor of 5th Embodiment, and is a figure explaining the reionization reaction of silver which occurs at the time of electrochemical measurement. It is a plane schematic diagram of the biomolecule sensor of 5th Embodiment. It is a cross-sectional schematic diagram of the biomolecule sensor of 5th Embodiment. It is a graph which shows a time-dependent change of the electrical potential difference at the time of connecting the reference electrode of an Example and a comparative example to the commercially available silver-silver chloride electrode. It is a graph which shows a time-dependent change of the electrical potential difference at the time of connecting the reference electrode of an Example and a comparative example to a gold electrode. It is a graph which shows the detection result of the nucleic acid by the nucleic acid sensor of an Example. It is a schematic diagram which shows the silver insolubilization reaction of an Example. It is a graph which shows the criminal change of the peak electric potential in the enzyme amount measurement test accompanying the silver insolubilization reaction of an Example.

  Hereinafter, a reference electrode according to an embodiment and an application example thereof will be described with reference to the drawings. The scope of the present invention is not limited to the following embodiments, and can be arbitrarily changed within the scope of the technical idea of the present invention. Moreover, in the following drawings, in order to make each structure easy to understand, the actual structure may be different from the scale or number of each structure.

<First Embodiment>
FIG. 1 is a cross-sectional view of an electrode 50 according to the first embodiment.
The electrode 50 includes a base layer 51, an Ag layer (silver layer, first member) 52 containing silver as a main component, an AgCl layer (silver chloride layer, second member) 53 containing silver chloride as a main component, SAM film (self-assembled monomolecular film) 54, and is formed in this order from the main surface 41 a side of the substrate 41.
In this specification, a generally known Ag / AgCl electrode is used for the Ag layer (first member) mainly composed of silver and the AgCl layer (second member) mainly composed of silver chloride. Silver and silver chloride may be contained to such an extent that the above functions are fulfilled.

  The substrate 41 is made of an insulating inorganic material such as glass. The shape of the electrode 50 is not particularly limited as long as it is a thin film, and the shape of the top view may be circular, elliptical, rectangular, triangular, polygonal, or indefinite.

  The base layer 51 is formed on the main surface 41a of the substrate 41 by a film forming method such as sputtering. The base layer 51 is not particularly limited as long as it is a layer containing a conductive substance. For example, the base layer 51 may be configured to include a metal, the carbon material described above, or the like. The base layer 51 may be made of, for example, gold or a gold compound. In this case, the base layer 51 has an effect that corrosion due to the biomolecule aqueous solution to be measured does not occur. When the base layer 51 is formed, a Cr layer for improving the bonding property between the substrate 41 and the base layer 51 may be provided in advance on the main surface 41 a of the substrate 41. Further, the base layer 51 may be omitted.

  The Ag layer 52 is formed on the surface of the base layer 51 on the main surface 41 a side of the substrate 41. Silver has a high affinity with the SAM film 54 described later. Therefore, the AgCl layer 53 can be provided on the surface of the Ag layer 52, and the SAM film 54 can be formed on the surface of the AgCl layer 53. The Ag layer 52 can be formed by sputtering. Alternatively, the Ag layer 52 may be formed by applying and fixing a silver paste to the base layer 51.

The AgCl layer 53 is formed so as to cover part or all of the surface of the Ag layer 52. Since the AgCl layer 53 is in contact with the aqueous biomolecule solution, the electrode 50 can be configured in which the potential at the solid-liquid interface with the aqueous solution is stabilized. This is considered to be because, when silver chloride comes into contact with the aqueous biomolecule solution, it is separated from Ag ions and Cl ions to reach an equilibrium state. The AgCl layer 53 can be formed by reacting the surface of the Ag layer 52 with a reagent such as iron chloride (FeCl ₃ ).
The AgCl layer (second member) 53 may be any layer as long as it is in contact with at least a part of the Ag layer 52 and may not be layered. For example, in this case, the AgCl layer 53 may be formed by bonding a thin film made of silver chloride separately prepared on the surface of the Ag layer 52.

  A SAM film (Self-Assembled Membrane) (SAM) 54 is formed on the surface of the AgCl layer 53 and covers the surface of the AgCl layer 53. The SAM film 54 is a coating layer (coating film) that uniformly covers the surface of the AgCl layer 53. The SAM film may cover the entire surface where the AgCl layer 53 is in contact with the solution to be measured. The SAM film may cover a part of the surface of the AgCl layer 53. The electrode 50 is in contact with the biomolecule solution to be measured in the SAM film 54.

The SAM film 54 is a monomolecular film made of organic molecules having high order in a self-organizing manner by utilizing the interaction between the surface of the AgCl layer 53 and organic compound molecules. The SAM film 54 is a monomolecular film made of organic molecules having high order in a self-organizing manner by utilizing the interaction between the surface of the AgCl layer 53 and organic compound molecules. The SAM film 54 includes a SAM film constituent material 54 a chemically adsorbed on the surface of the AgCl layer 53.
Note that a layer formed of a different material (for example, a buffer layer or a buffer layer) may be formed at least partially between the SAM film 54 and the AgCl layer 53. In addition, layers formed of different substances may be sandwiched between SAM films. In addition, the first SAM film and the second SAM film may be formed, and a layer formed of a different substance may exist between the first SAM film and the second SAM film. . Note that the first SAM film and the second SAM film may be made of different constituent materials, or may be made of the same constituent material.

Examples of the SAM film constituent material 54a include alkanethiol derivatives. However, the composition of the SAM film 54 is not limited to this, and the composition is not limited as long as it covers the surface of the AgCl layer 53 closely. In the present embodiment, mercaptohexanol is used as the SAM film constituent material 54a among the alkanethiol derivatives.
Examples of the alkanethiol derivative include compounds represented by the following general formulas (11) to (16).

[In said general formula (11)-(16), n and m are 1-100 each independently. ]
For example, n and m are each independently 1-30, or are each independently 1-20. For example, in this embodiment, the compound represented by the said General formula (14) is mentioned among the compounds represented by the said General formula (11)-(16).
In the general formula (14), for example, n is 1 to 15, or 1 to 10. Such compounds include n = 6 mercaptohexanol.

  The film thickness of the SAM film 54 depends on the type of the SAM film constituent material 54a, but is, for example, 1 nm or more and 10 nm or less. By forming a film having such a thickness on the surface of the AgCl layer 53, elution of Cl ions from the surface of the AgCl layer 53 into the biomolecule solution can be appropriately suppressed.

  FIG. 2 is a schematic diagram for explaining the bonding with the AgCl layer 53 when an alkanethiol derivative is used as the SAM film constituent material 54a. Sulfur contained in the thiol group of the alkanethiol derivative has high binding affinity with noble metals such as silver and gold. On the surface of the AgCl layer 53, silver chloride Cl ions are exposed. Under such circumstances, when a sulfur-containing compound of a thiol group of an alkanethiol derivative is allowed to act, Cl ions are replaced with sulfur, and silver and sulfur are bonded. Thus, sulfur binds to the surface of the AgCl layer 53 to immobilize the alkanethiol derivative. Note that in the portion where the surface of the Ag layer 52 is exposed from the AgCl layer 53, the sulfur-containing compound of the thiol group of the alkanethiol derivative and silver act to bond sulfur directly to silver.

  The SAM film 54 can be formed by applying a solution in which the SAM film constituent material 54a is dissolved to the surface of the AgCl layer 53 and drying it. As a method of applying the SAM film constituent material solution to the AgCl layer 53, a method of applying the solution by spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, or the like, a dropping method, spraying Examples thereof include a method of applying by a method, a vapor deposition method, a casting method, a dipping method and the like. As a solvent for dissolving the SAM film constituent material 54a, water, alcohol, or a mixed solvent thereof is used.

The fact that the SAM film 54 is immobilized on the surface of the AgCl layer 53 can be confirmed by performing XPS analysis (X-ray photoelectron spectroscopy, X-ray photoelectron spectroscopy) on the surface of the AgCl layer 53. .
3 and 4 are graphs showing the results of XPS analysis of the surface of the AgCl layer 53 before and after forming the SAM film 54, respectively. Prior to the formation of the SAM film 54 (FIG. 3), a peak derived from the undetected sulfur electrons is detected after the formation of the SAM film 54 (FIG. 4).

  The SAM film 54 suppresses the transfer of Cl ions between the biomolecule aqueous solution and the AgCl layer 53 on the surface of the electrode 50 as compared with the case where the SAM film 54 is not provided. The provision of the SAM film 54 makes it difficult for Cl ions to elute from the AgCl layer 53. For this reason, the electrode 50 provided with the SAM film 54 hardly affects the Cl ion concentration of the target biomolecule aqueous solution, and enables more stable measurement. In addition, by providing the electrode with the SAM film 54, it takes a long time until the Cl ions are completely eluted from the AgCl layer 53, so that the life of the electrode 50 can be extended.

  FIG. 5 and FIG. 6 are images obtained by imaging the surface of the AgCl layer 53 before and after the formation of the SAM film 54 under the same conditions using an SEM (Scanning Electron Microscope). Comparing before formation (FIG. 5) and after formation (FIG. 6) of the SAM film 54, it can be seen that the resolution of the SEM image after formation is poor. This is a phenomenon called charge-up that occurs because the surface of the target AgCl layer 53 is charged by SEM imaging. As described above, since charging is generated on the surface of the AgCl layer 53, it can be confirmed that formation of the SAM film 54 suppresses charge transfer on the surface of the AgCl layer 53. From this, it can be said that the AgCl layer 53 is in a semi-insulating state by the SAM film 54. Here, the semi-insulating state means a state in which electrical exchange with the solution (more specifically, elution of Cl ions) is suppressed. Therefore, even if the AgCl layer 53 and the solution are locally in a conductive state, the state where electrical exchange is suppressed as a whole is included in the semi-insulated state. On the surface of the AgCl layer 53, the gap between the crystal grains has an intricate shape, which is considered to be a region where the SAM film 54 is difficult to be formed. In the SAM film 54, it is considered that the AgCl layer 53 can be in a semi-insulating state because the AgCl layer 53 and the solution allow electrical exchange in such a local region.

The electrode 50 can be used as a reference electrode as an example. When the electrode 50 is used as a reference electrode, the exchange of Cl ions between the AgCl layer 53 and the aqueous solution is suppressed by the SAM film 54, and Cl ions are less likely to be eluted from the AgCl layer 53. In general, it is known that a phenomenon in which the potential at the solid-liquid interface of the reference electrode becomes unstable (referred to as potential drift) is caused by an increase or decrease in ion concentration in the aqueous biomolecule solution. In the electrode 50 of the present embodiment, the elution of Cl ions from the AgCl layer 53 is suppressed, so that the ion concentration in the aqueous biomolecule solution hardly changes and the potential drift of the electrode 50 can be suppressed. In addition, since the electrode 50 of the present embodiment has little change in the Cl ion concentration of the aqueous biomolecule solution, it is not necessary to wait until the ion concentration becomes stable, and measurement can be started immediately.
Moreover, since the electrode 50 of this embodiment has little elution of Cl ions, it takes a long time until the Cl ions are completely eluted from the AgCl layer 53 and can have a long life. Furthermore, according to the electrode 50 of the present embodiment, since the elution of Cl ions from the AgCl layer 53 occurs from the entire surface of the AgCl layer 53, local consumption of Cl ions hardly occurs. Therefore, the electrode 50 of the present embodiment can further increase the life.
In addition, non-specific adsorption to the electrode 50 is suppressed by the SAM film 54, which can further contribute to potential stability. According to the present embodiment, it is possible to provide a reference electrode that enables stable potential measurement.

  The electrode 50 may be used as a working electrode. When the electrode 50 is used as a reference electrode, the measurement potential can be stabilized by suppressing nonspecific adsorption on the surface of the electrode 50 by the SAM film 54.

  The electrode 50 of this embodiment can be applied as an electrochemical sensor. In particular, the electrode 50 can be applied as a biosensor for detecting a biomolecule as described in the following embodiments. When the electrode 50 is applied as a biosensor, examples of biomolecules to be detected include nucleic acids, DNA, RNA, proteins, peptides, extracellular vesicles, antibodies, and antigens.

Second Embodiment
FIG. 7 is a schematic cross-sectional view of the nucleic acid sensor 40 of the second embodiment. For example, the nucleic acid sensor 40 includes a substrate (electrode substrate) 41, a reference electrode 50 formed on the main surface 41 a of the substrate 41, and a pair of working electrodes 60. The nucleic acid sensor 40 includes a potential measurement unit (not shown) that is electrically connected to the reference electrode 50 and the working electrode 60. The potential measuring unit measures a potential difference between the reference electrode 50 and the working electrode 60. The nucleic acid sensor 40 brings the reference electrode 50 and the working electrode 60 into contact with a solution (biomolecule aqueous solution) to be measured, whereby a target nucleic acid (eg, microRNA (hereinafter referred to as miRNA)) contained in the biomolecule aqueous solution. ).
The reference electrode 50 of this embodiment has the same mode as the electrode 50 of the first embodiment. In addition, the same reference numerals are given to components having the same aspects as those in the first embodiment, and the description thereof is omitted.

(Working electrode)
The working electrode 60 includes a base layer (working electrode side base layer) 61, a SAM film (working electrode side SAM film) 62 and a capture probe 70 formed on the surface of the base layer 61.

  The base layer 61 is formed on the main surface 41a of the substrate 41 by a film forming method such as sputtering. Note that the base layer 61 of the working electrode 60 may be made of the same material as the base layer 51 of the reference electrode 50. As a result, the base layers 51 and 61 can be formed at the same time, and the manufacturing cost can be reduced.

  The SAM film 62 covers the surface of the base layer 61 and suppresses nonspecific adsorption from occurring on the surface of the base layer 61. The SAM film 62 of the working electrode 60 has the same composition as the SAM film 54 of the reference electrode 50, and both are made of the SAM film constituent material 54a having the same composition. As a result, the SAM film 62 of the working electrode 60 can be formed simultaneously with the SAM film 54 of the reference electrode 50, and the manufacturing cost can be reduced. In the present embodiment, the SAM film 62 of the working electrode 60 is made of an alkanethiol derivative (for example, mercaptohexanol) chemisorbed on the surface of the base layer 61. Note that the sulfur contained in the thiol group of the alkanethiol derivative acts stably with gold and binds stably.

The capture probe 70 constitutes a capture unit 75 that can react with a biomolecule (nucleic acid, miRA) to be detected. The working electrode 60 has a capturing part on the surface.
The capture probe 70 has a sequence capable of hybridizing with the nucleic acid to be detected. In the present specification, “capable of hybridizing” means that a part of the capture probe 70 used hybridizes to a target nucleic acid (target miRNA) under stringent conditions, and a nucleic acid other than the target nucleic acid (target miRNA). It means that the molecule does not hybridize or is difficult to hybridize. “Stringent conditions” include, for example, conditions described in Molecular Cloning-A LABORATORY MANUAL 2nd EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press).

  Next, two types of capture probes (first capture probe 70A and second capture probe 70B) that can be employed as the capture probe 70 will be described with reference to FIGS. The capture probe 70 is not limited to the one described here as long as it has a sequence capable of hybridizing with the nucleic acid (miRNA) to be detected.

  FIG. 8 is a schematic diagram of the first capture probe 70A. The first capture probe 70 </ b> A includes a first portion 71, a stem portion 73, and a spacer 74. The first portion 71 is composed of a sequence capable of hybridizing with miRNA. The stem portion 73 has a loop structure 73b and a double strand 73a connected to the loop structure 73b.

  The length (eg, the number of bases) of the stem portion 73 is not particularly limited, but may be about 8 to 15 bases as a guide in consideration of the length capable of stably forming the double strand 73a. The stem portion 73 does not need to form the base pair of the double strand 73a as a whole, but the stem portion 73 is adjacent to the first portion 71, and miRNA is hybridized to the first portion 71. Therefore, it is preferable that at least the base adjacent to the first portion 71 in the stem portion 73 forms a base pair.

  FIG. 9 is a schematic diagram of the second capture probe 70B. The second capture probe 70B is different from the first capture probe 70A in that it does not have the stem portion 73.

  The capture probes 70 (the first capture probe 70A and the second capture probe 70B) are spacers at the ends in order to ensure molecular freedom for hybridizing the miRNA while being fixed to the base layer 61. 74. The length of the spacer 74 is not particularly limited, but is, for example, 3 bases to 50 bases, or 5 bases to 25 bases. However, the base used for the spacer 74 can be replaced with a linker such as PEG having the same length and softness. In such a case, the number of bases used for the spacer 74 may be 0 bases. Further, the SAM film constituent material may be used as the spacer 74 by configuring the capture probe 70 at the tip of the SAM film constituent material constituting the SAM film 62.

  The capture probe 70 may be DNA or RNA, and is not limited to natural and non-natural, as long as it has a function similar to DNA or RNA, but is not limited to PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), BNA (Bridged Nucleic). Acid) or other artificial nucleic acid may be included. Compared to DNA and RNA, the capture probe 70 has a high affinity with the target miRNA, is not easily recognized by a DNA degrading enzyme or an RNA degrading enzyme, and can be a substrate for a DNA ligase such as T4 DNA ligase. Preferably it contains BNA.

  The nucleic acid sensor 40 has a potential difference measurement unit (not shown) that is electrically connected to the reference electrode 50 and the working electrode 60. The potential difference measurement unit measures the potential difference between the reference electrode 50 and the working electrode 60, thereby causing the working probe 60 to capture the target microRNA (miRNA) and the solution ( Changes in potential (changes in surface charge density) at the solid / liquid interface (also referred to as solid-liquid interface) with the liquid are detected. Thereby, it can be easily determined whether or not the target miRNA is contained in the aqueous biomolecule solution. The nucleic acid sensor 40 detects the target nucleic acid by measuring a potential change associated with the hybridization between the capture probe 70 and the target nucleic acid.

  According to the reference electrode 50 of the present embodiment, the surface of the AgCl layer 53 is covered with the SAM film 54. Thereby, the exchange of Cl ions between the AgCl layer 53 and the aqueous biomolecule solution is suppressed, and the elution of Cl ions from the AgCl layer 53 hardly occurs. In general, it is known that a phenomenon in which the potential at the solid-liquid interface of the reference electrode becomes unstable (referred to as potential drift) occurs when the ion concentration in the aqueous biomolecule solution increases or decreases. By suppressing the elution of Cl ions from the AgCl layer 53, the ion concentration in the aqueous biomolecule solution hardly changes, and the potential drift of the reference electrode 50 can be reduced. In addition, since there is little change in the Cl ion concentration of the aqueous biomolecule solution, it is not necessary to wait until the ion concentration is stabilized, and measurement can be started immediately.

  In addition, since the reference electrode 50 of this embodiment has little elution of Cl ions, it takes a long time until the Cl ions are completely eluted from the AgCl layer 53 and can have a long life. Furthermore, according to the reference electrode 50 of this embodiment, since the elution of Cl ions from the AgCl layer 53 occurs from the entire surface of the AgCl layer 53, local consumption of Cl ions hardly occurs. Therefore, the reference electrode 50 of the present embodiment can further increase the life.

  The nucleic acid sensor 40 of this embodiment can be used, for example, for determining whether or not a subject has cancer cells. Abnormal cells such as cancer cells express specific proteins and nucleic acids such as miRNA inside the cell membrane. Because exosomes secreted into body fluids (blood, urine, saliva, etc.) express miRNA derived from the secretory cell, analyze biomolecules that exist inside the membrane of exosomes in body fluids Thus, it is possible to investigate abnormalities in the living body. Therefore, the presence or absence of cancer cells can be evaluated by comparing the expression level of a predetermined miRNA in a cancer cell-derived exosome with the expression level of miRNA in a exosome derived from a subject. In addition, miRNA is not limited to what exists in an exosome. For example, miRNAs present in body fluids such as blood, urine, and saliva may be directly detected. It is also possible to detect and diagnose other diseases by comparing the expression levels of miRNA.

  In the present embodiment, the reference electrode 50 is formed on the substrate 41. The Ag layer 52 as the first member is a layer formed on the surface of the substrate 41 via the base layer 51. Similarly, the AgCl layer 53 as the second member is a layer formed on the surface of the base layer 51. However, the first member 52 and the second member 53 are not limited to these forms. The first member 52 and the second member 53 may be independent members such as rods and plates that are electrically connected to each other. In addition, a layered second member may be formed on the surface of the rod-shaped first member 52. As long as the first member 52 and the second member 53 are electrically connected to each other and have a surface that is exposed to form the SAM film 54 on the surface of the second member 53, any type of member can be used. The form is not limited.

<Third Embodiment>
Next, the fluid device 21 of the third embodiment will be described. The fluidic device 21 of this embodiment is a device that detects a biomolecule (eg, miRNA) contained in an exosome in a sample.
FIG. 10 is an exploded view of the fluidic device 21. As will be described later, the fluidic device 21 includes the nucleic acid sensor 40 of the second embodiment. Moreover, about the component of the same aspect as 2nd Embodiment, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  The fluid device 21 includes a flow path base material 30, an electrode substrate 41, an insulating film 24, and a circuit substrate 25. Further, the flow path base material 30, the electrode substrate 41, the insulating film 24, and the circuit board 25 are laminated in this order in the vertical direction.

(Channel substrate)
The channel substrate 30 is a plate material made of PDMS (Poly Dimethyl Siloxane) and has an upper surface 30a and a lower surface 30b. The flow path base 30 has a pair of introduction through holes 32 and one discharge through hole 31 that penetrate from the upper surface 30a to the lower surface 30b. On the lower surface 30 b of the flow path base material 30, a groove-shaped flow path 33 extending from each introduction through hole 32 and connected to the discharge through hole 31 is provided.

  The lower surface 30 b of the flow path base material 30 is covered with the main surface 41 a of the electrode substrate 41. An introduction tube that supplies a biomolecule solution containing a biomolecule to be detected (for example, miRNA) to the flow path 33 can be connected to the pair of introduction through holes 32. In addition, a discharge tube that discharges the biomolecule solution that has passed through the flow path 33 from the introduction tube can be connected to the discharge through hole 31. The biomolecule solution supplied from the introduction tube flows through the flow path 33 from the introduction through hole 32 and reaches the discharge through hole 31. The biomolecule solution discharged from the discharge tube is supplied again from the introduction tube to the flow path 33 through a pump connected to the outside. Thus, the biomolecule solution circulates through the flow path 33.

A method for manufacturing the flow path base material 30 will be described.
First, a flat glass substrate is prepared, and a mold for forming a minute flow path structure is formed on the glass substrate. An electrode substrate 41 may be used as the glass substrate. A thick film photoresist is coated on a glass substrate and baked. Next, after exposure using a photomask having a pattern corresponding to the flow path, baking is performed again for 10 minutes. Subsequently, it developed in the developing solution for thick film photoresists, and dried. Through the above steps, a mold for forming the flow channel structure is produced.
Next, the flow path base material 30 is produced using the mold described above. First, a liquid mixture of a PDMS precursor and a curing agent is uniformly applied on a glass substrate on which a mold is formed, and after the bubbles are removed, it is cured. Subsequently, the base material of PDMS provided with the flow path 33 can be molded by releasing the cured PDMS from the glass substrate. Furthermore, the flow path base material can be manufactured by mechanically processing the introduction through hole 32 and the discharge through hole 31.

(Electrode substrate)
On the main surface 41a of the electrode substrate 41, a reference electrode 50 and a pair of working electrodes 60 are provided. The electrode substrate 41, the reference electrode 50, and the pair of working electrodes 60 have the same configuration as that of the second embodiment, and these constitute the nucleic acid sensor 40. The reference electrode 50 includes a base layer 51, an Ag layer 52 mainly composed of silver, an AgCl layer 53 mainly composed of silver chloride, and a SAM film (self-assembled monomolecular film) 54. The working electrode 60 includes a base layer (working electrode side base layer) 61, a SAM film (working electrode side SAM film) 62 formed on the surface of the base layer 61, and a capture probe 70.

  The electrode substrate 41 is overlaid on the flow path base material 30. Thereby, at least a part of the reference electrode 50 overlaps with the discharge through hole 31 of the flow path base material 30. Further, at least a part of the pair of working electrodes 60 is disposed in different flow paths 33.

(Insulating film)
The insulating film 24 is made of polyimide, for example. The insulating film 24 prevents an unexpected short circuit in the control circuit 25 a provided on the circuit board 25.

(Circuit board)
The circuit board 25 includes a control circuit 25a that controls the electrodes (the working electrode 60 and the reference electrode 50) of the electrode substrate 41. The control circuit 25 a is electrically connected to the pair of working electrode 60 and reference electrode 50. In the present embodiment, the control circuit 25a and the electrodes (working electrode 60 and reference electrode 50) are connected by a lead wire (not shown). In addition, a through hole may be provided in the electrode substrate 41 and the insulating film 24 to connect the control circuit 25a and each electrode.

(Function and effect)
The fluidic device 21 of the present embodiment can detect a nucleic acid (for example, miRNA) by measuring a potential change accompanying the hybridization of the capture probe 70 and miRNA on the working electrode 60 of the electrode substrate 41 on the circuit board 25. .
According to this embodiment, nucleic acid can be detected with higher accuracy by circulating a biomolecule solution to be measured by a pump (not shown).
In the present embodiment, the detection target is miRNA. However, even other biomolecules (for example, nucleic acids such as DNA and RNA) can be detected with the same configuration.

<Fourth embodiment>
Next, the fluidic device 1 of 4th Embodiment is demonstrated. The fluidic device 1 of the present embodiment is a device that detects a biomolecule (eg, miRNA) contained in an exosome in a sample.
FIG. 11 is a schematic diagram of the fluidic device 1. The fluidic device is, for example, a microfluidic device (microTAS), a millifluidic device (milliTAS), or the like. The fluidic device 1 includes the nucleic acid sensor 40 of the second embodiment in the biomolecule detection unit 4 as described later.

The fluidic device 1 includes an exosome purification unit 2 having a layer modified with a compound having a hydrophobic chain and a hydrophilic chain, a biomolecule purification unit (target purification unit) 3, a biomolecule detection unit 4, and an exosome purification. A first flow path 5 connecting the section 2 and the biomolecule purification section 3, and a second flow path 6 connecting the biomolecule purification section 3 and the biomolecule detection section 4.
In the present embodiment, the first flow path 5 is a flow path for sending an exosome crushing liquid containing biomolecules from the exosome purification section 2 to the biomolecule purification section 3, and the second flow path 6 is a purification path. This is a flow path for feeding the solution containing the biomolecules to the biomolecule detection unit 4.
Further, for example, the fluid device 1 of the present embodiment is a device that introduces a sample, crushes exosomes, purifies a biomolecule, and detects the biomolecule.
Furthermore, the fluid device 1 includes a first waste liquid tank 7, a second waste liquid tank 8, a third waste liquid tank 9, and a third flow path 10 that connects the first waste liquid tank 7 and the exosome purification unit 2. And a fourth flow path 11 that connects the second waste liquid tank 8 and the biomolecule purification unit 3, and a fifth flow path 12 that connects the third waste liquid tank 9 and the biomolecule detection unit 4.

(Exosome Purification Department)
The exosome purification unit 2 performs exosome immobilization and exosome crushing, and includes an exosome immobilization unit 2d having an inlet and a layer modified with a compound having a hydrophobic chain and a hydrophilic chain. The exosome purification unit 2 preferably includes an inlet for each reagent to be introduced. That is, the exosome purification unit 2 preferably includes a sample introduction inlet 2b and a crushing liquid introduction inlet 2c, and more preferably includes a washing liquid introduction inlet 2a.

The compound having a hydrophobic chain and a hydrophilic chain in the exosome fixing part 2d is a compound having a hydrophobic chain for binding to the lipid bilayer and a hydrophilic chain for dissolving the lipid chain. By using such a compound, an exosome having a lipid bilayer can be fixed on the exosome fixing part 2d.
In the present specification, “immobilizing an exosome on the exosome fixing part 2d” also means adsorbing the exosome to the exosome fixing part. Isolation of exosomes from the sample is possible.

The hydrophobic chain may be single chain or double chain, and examples thereof include a saturated or unsaturated hydrocarbon group which may have a substituent.
As the saturated or unsaturated hydrocarbon group, a linear or branched alkyl group or alkenyl group having 6 to 24 carbon atoms is preferable, and a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, Dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, stearyl group (octadecyl group), nonadecyl group, icosyl group, heicosyl group, docosyl group, tricosyl group, tetracosyl group, myristolyl group, palmitoleyl group Oleyl group, linoleyl group, linoleyl group, ricinoleyl group, isostearyl group and the like. Among these, a myristol group, a palmitoleyl group, an oleyl group, a linoleyl group, and a linoleyl group are preferable, and an oleyl group is more preferable.

  Examples of the hydrophilic chain include protein, oligopeptide, polypeptide, polyacrylamide, polyethylene glycol (PEG), dextran, and the like, and PEG is preferable. Such hydrophilic chains are preferably chemically modified for bonding to the substrate, more preferably have an active ester group, and particularly preferably an N-hydroxysuccinimide group.

  That is, as a compound having a hydrophobic chain and a hydrophilic chain, a lipid-PEG derivative is preferable. The lipid-PEG derivative is called BAM (Biocompatible anchor for membrane). Examples of BAM include a compound represented by the following formula (1).

[Wherein n is an integer of 1 or more. ]
Examples of the substrate used as the layer of the exosome fixing part 2d include a glass substrate, a silicon substrate, a polymer substrate, and a metal substrate. The substrate may be bonded via a substance that binds to a hydrophilic chain of a compound having a hydrophobic chain and a hydrophilic chain. Examples of such a substance include an amino group, a carboxyl group, a thiol group, a hydroxyl group, and an aldehyde group. And 3-aminopropyltriethoxysilane is preferable.

  The liquid in the fluid device 1 of this embodiment is driven by an external suction pump (not shown), and the flow of the liquid is controlled by opening and closing a valve. The valve is, for example, a pneumatic valve, and opening / closing of the valve is driven and controlled by an external pneumatic device connected to the fluid device 1.

In the analysis of exosomes, first, in the exosome purification section described above, a sample is injected into the sample introduction inlet 2b, the valve 2f of the flow path 2i is opened, and the sample is introduced into the exosome fixing section 2d by suction.
The sample amount used for the analysis is, for example, from several μL to several mL, for example, about 1 mL.

The sample is obtained from the environment surrounding the cell to be detected and is not particularly limited as long as it contains the exosome secreted by the cell. Blood, urine, breast milk, bronchoalveolar lavage fluid, amniotic fluid, Examples include malignant exudates and saliva. Among these, blood or urine that can easily detect exosomes is preferable. Furthermore, in blood, plasma is preferable because of easy detection of exosomes.
Such a sample also includes a cell culture medium containing exosomes secreted by cultured cells.

Examples of cells to be detected include cancer cells, mast cells, dendritic cells, reticulocytes, epithelial cells, B cells, and nerve cells that are known to produce exosomes.
The sample may be prepared by ultracentrifugation, ultrafiltration, continuous flow electrophoresis, filtration using a size filter, gel filtration chromatography, or the like, but in this embodiment, the exosome fixing part 2d. Since the affinity between a compound having a hydrophobic chain and a hydrophilic chain in exosome and exosome is very high, the sample itself may not be prepared.

  From the viewpoint of specifically binding the exosome to the exosome fixing part 2d, it is preferable to provide a nonspecific adsorption suppressing part in the exosome fixing part 2d. As an example, after the substrate is modified with a compound having a hydrophobic chain and a hydrophilic chain, a portion where the compound having the hydrophobic chain and the hydrophilic chain is not modified is treated with a compound having a hydrophilic group such as PEG. Can be mentioned.

The exosome in the sample introduced into the exosome fixing part 2d is captured by the compound having the hydrophobic chain and the hydrophilic chain described above. Since the affinity between the exosome and the compound having a hydrophobic chain and a hydrophilic chain is very high, it is not necessary to leave the sample in the exosome fixing part 2d, and at the same time as the sample continuously passes over the exosome fixing part 2d. The exosome in the sample is captured on the exosome fixing part 2d.
As an example, the suction pressure during exosome capture is 1 kPa to 30 kPa, and the time required for capture is about 15 seconds. The solution that has passed through the exosome fixing part 2d is sent to the first waste liquid tank 7 through the third flow path 10 via the valve 10a.

  In the fluid device 1 of the present embodiment, it is preferable to design the ceiling height of the exosome fixing part 2d low. Thereby, the opportunity of contact with the compound which has a hydrophobic chain | strand and a hydrophilic chain | strand and exosome can be increased, and the capture efficiency of exosome can be improved.

In addition to exosomes, extracellular vesicles such as microvesicles and apoptotic bodies are contained in blood, and these extracellular vesicles may be fixed to the exosome fixing part 2d. From the viewpoint of removing these extracellular vesicles from the exosome fixing part 2d, it is preferable to wash the exosomes on the exosome fixing part 2d.
As an example, the valve 2e on the flow path 2h is opened, the cleaning liquid is injected into the cleaning liquid introduction inlet 2a, and is introduced into the exosome fixing part 2d.
In this embodiment, since the bond between the layer modified with the compound having a hydrophobic chain and a hydrophilic chain and the exosome is strong, the flow rate can be adjusted quickly, and washing in a short time is possible. As an example, the cleaning is performed by feeding 500 μL of PBS cleaning solution at a suction pressure of 1 kPa to 30 kPa for about 15 seconds. The waste liquid that has passed through the exosome fixing part 2d is sent to the first waste liquid tank 7 through the third flow path 10 via the valve 10a.

Subsequently, the exosome fixed to the exosome fixing part 2d is crushed. The valve 2g on the flow path 2j is opened, the crushing liquid is injected into the inlet 2c for crushing liquid introduction, and the crushing liquid is introduced into the exosome fixing part 2d by suction. Examples of the crushing liquid include conventionally known ones used for cell lysis.
When the disrupted liquid passes through the exosome fixing part 2d, the exosome captured on the exosome fixing part 2d is crushed, and biomolecules (nucleic acid, particularly miRNA) encapsulated in the exosome are released.
As an example, the suction pressure at the time of exosome crushing is 1 kPa to 30 kPa, and the time required for crushing is about 30 seconds. The waste liquid that has passed through the exosome fixing part 2d is sent to the first waste liquid tank 7 through the third flow path 10 via the valve 10a. The biomolecule released from the exosome is sent to the biomolecule purification unit (object purification unit) 3 through the first flow path 5 via the valve 5a.

(Biomolecule Purification Department (Object Purification Department))
The biomolecule purification unit extracts biomolecules in the sample. The biomolecule purification unit 3 preferably includes a biomolecule recovery liquid introduction inlet 3b and a biomolecule fixing unit 3c, and more preferably includes a biomolecule cleaning liquid introduction inlet 3a.
The biomolecule immobilization part 3c is not particularly limited as long as it can immobilize a biomolecule (nucleic acid), but a silica membrane that immobilizes a nucleic acid is an example.
The exosome holds a nucleic acid derived from a secretory cell. Examples of the nucleic acid include miRNA. In recent years, it has been reported that miRNA, a short non-coding RNA, regulates gene expression in vivo, and the relationship between abnormal expression of miRNA and various diseases such as cancer has been clarified. It's getting on.
When the exosome crushing liquid passes through the biomolecule fixing part 3c, the biomolecule (nucleic acid) is captured on the biomolecule fixing part 3c.
As an example, the suction pressure at the time of exosome disruption liquid feeding is 50 kPa-70 kPa, and the time required for liquid feeding is about 1 minute. The waste liquid that has passed through the biomolecule fixing part 3c is sent to the second waste liquid tank 8 through the fourth channel 11 via the valve 11a.

After immobilizing the biomolecule to the biomolecule immobilization part 3c, it is preferable to wash the biomolecule immobilization part 3c and remove impurities other than the target biomolecule. The valve 3d on the flow path 3e is opened, the cleaning liquid is injected into the biomolecule cleaning liquid introduction inlet 3a, and the cleaning liquid is introduced into the biomolecule fixing part 3c by suction. Examples of the cleaning liquid include about 70% to 80% ethanol.
As an example, the amount of cleaning liquid used at the time of cleaning is about 1 mL, the suction pressure is 50 kPa to 70 kPa, and the time required for feeding the cleaning liquid is about 1 minute. The waste liquid that has passed through the biomolecule fixing part 3c is sent to the second waste liquid tank 8 through the fourth channel 11 via the valve 11a.

  It is preferable to dry the biomolecule fixing part 3c after washing the biomolecule fixing part 3c. This is to prevent the biomolecule cleaning liquid from being brought into the biomolecule detection unit. The biomolecule fixing part 3c is dried by sucking air from the biomolecule cleaning liquid introducing inlet 3a and passing it through the biomolecule fixing part 3c. As an example, the suction pressure when drying the biomolecule fixing part 3c is 50 kPa to 70 kPa, and the time required for drying is about 2 minutes.

  Next, the biomolecule fixed to the biomolecule fixing part 3c is eluted. In order to improve the recovery rate of biomolecules, it is preferable to hold the biomolecule recovery liquid for a certain period of time after introducing the biomolecule recovery liquid into the biomolecule fixing part 3c. The valve 3f of the flow path 3g is opened, the biomolecule recovery liquid is injected into the biomolecule recovery liquid introduction inlet 3b, and the biomolecule recovery liquid is introduced into the biomolecule fixing part 3c. As an example, the biomolecule recovery liquid is RNase-free water, the usage amount of the recovery liquid is 30 μL, the recovery liquid is aspirated at a suction pressure of 50 kPa to 70 kPa, and the recovery liquid reaches the biomolecule fixing part 3c. Suction is stopped at the time and held for about 3 minutes.

  Next, the biomolecule is recovered from the biomolecule fixing part 3c. As an example, the recovered liquid is recovered over 30 seconds at a suction pressure of 50 kPa to 70 kPa. The recovered biomolecule passes through the second flow path 6 and is sent to the biomolecule detection unit 4. As an example, the suction pressure of the biomolecule to the biomolecule detection unit 4 is 6 kPa or less, and the liquid is fed over about 30 seconds.

(Biomolecule detection part)
The biomolecule detection unit 4 includes a circulation type mixing unit 4c having an annular circulation channel 4f and a nucleic acid sensor 40 provided in the path of the circulation channel 4f.

  As described in the second embodiment with reference to FIG. 7, the nucleic acid sensor 40 includes a substrate 41, a reference electrode 50 provided on the substrate 41, and a working electrode 60. The reference electrode 50 includes a base layer 51, an Ag layer 52 mainly composed of silver, an AgCl layer 53 mainly composed of silver chloride, and a SAM film (self-assembled monomolecular film) 54. The working electrode 60 includes a base layer (working electrode side base layer) 61, a SAM film (working electrode side SAM film) 62 formed on the surface of the base layer 61, and a capture probe 70. The biomolecule detection unit 4 detects a nucleic acid by measuring a potential change accompanying hybridization between a capture probe 70 fixed to the working electrode 60 and a target nucleic acid (for example, miRNA).

  The circulation type mixing unit 4c has a circulation channel 4f provided with a pump valve (not shown) in the channel. A second flow path 6 extending from the biomolecule purification unit 3 and a fifth flow path 12 extending to the third waste liquid tank 9 are connected to the circulation flow path 4f. In addition, a cleaning liquid inlet 4b is connected to the circulation channel 4f via a valve 4e.

  When the biomolecule is sent to the biomolecule detection unit 4, a pump valve (not shown) is operated to circulate and mix the biomolecule aqueous solution in the circulation channel 4f. As an example, opening and closing of a pump valve (not shown) is continued for about 10 minutes. By circulating the liquid, the capture probe 70 (see FIG. 1) and the target nucleic acid hybridize in the nucleic acid sensor 40 efficiently in a short time.

  Next, the valve 4 e is opened, the cleaning liquid is injected into the cleaning liquid introduction inlet 4 b and introduced into the nucleic acid sensor 40. Thereby, the nonspecific adsorbate adhering to the working electrode 60 of the nucleic acid sensor 40 can be removed. The waste liquid that has washed the nucleic acid sensor 40 passes through the fifth flow path 12 via the valve 12 a and is sent to the third waste liquid tank 9.

  Next, the nucleic acid is detected by measuring the potential of the working electrode 60 with respect to the reference electrode 50 in the nucleic acid sensor 40.

  According to the fluidic device of the present embodiment, the action of extracting and detecting nucleic acids from exosomes can be quickly performed in the same device.

<Fifth Embodiment>
Next, the biomolecule sensor 140 of 5th Embodiment is demonstrated. The biomolecule sensor 140 of this embodiment reacts a biomolecule with a specific substance exhibiting a selective interaction, and performs an insolubilization reaction in correlation with the abundance of the biomolecule contained in the solution to be measured. Thus, the soluble substance is converted into an insoluble substance and deposited on the working electrode, and the deposited insoluble substance is electrically analyzed.

  12 and 13 are reaction schematic diagrams of the analysis system in the biomolecule sensor 140 of the present embodiment. FIG. 14 is a schematic plan view of the biomolecule sensor 140, and FIG. 15 is a schematic cross-sectional view of the biomolecule sensor 140.

(Biomolecule detection method)
In the analysis systems shown in FIG. 12 and FIG. 13, the antigen 103 is used as a biomolecule of a substance to be analyzed, and an immune complex formation reaction based on an antigen-antibody reaction (sandwich method) is used on the magnetic fine particle 105 as a selective interaction. As one of them, an ALP-labeled antibody 104 obtained by labeling an antibody that specifically reacts with the antigen with an enzyme [for example, alkaline phosphatase (ALP)] is used.
In this analysis system, the following reaction formula 1 is used as the enzyme reaction by the labeling enzyme, and the reaction formula 2 is used as the insolubilization reaction. 12 and 13, pAPP means p-aminophenyl phosphate, pAP means p-aminophenol, pQI means p-quinoneimine, and pAPP is an ALP substrate. Further, “(ALP)” in Reaction Scheme 1 indicates that ALP is involved as a catalyst in Reaction Scheme 1.
Furthermore, as an electrical analysis method, an electrode portion including a working electrode (working electrode) 160, a counter electrode (counter electrode) 170, and a reference electrode (reference electrode) 150 is used.

In the analysis systems shown in FIGS. 12 and 13, an antibody 102 against a biomolecule (antigen) to be analyzed is immobilized on a magnetic microparticle 105 in advance. The working electrode 160 constituting the electrode unit functions as a sensing unit. First, when a test sample containing a biomolecule to be analyzed (antigen 103), immobilized microparticles, and ALP-labeled antibody 104 are reacted in a reaction part, a complex of immobilized antibody microparticles / antigen / ALP-labeled antibody is formed. Is done. The amount of complex formation correlates with the amount of biomolecules present in the test sample. After the complex is formed, B / F separation is performed with an appropriate cleaning solution, the B / F is separated and moved to the sensing unit, and magnetic particles are collected on the sensing unit by a magnet (not shown) on the back of the sensing unit. In addition, the antibody or antigen-bound microparticles may be immobilized in advance on the sensing part, and after the formation of the antibody / antigen / ALP labeled antibody complex on the sensing part, B / F separation may be performed with an appropriate washing solution. Is also possible. After collecting the magnetic fine particles on which the antibody / antigen / ALP labeled antibody complex is immobilized on the sensing part, or at the same time as collecting the magnetic fine particles on the sensing part while moving to the sensing part, p- which is a substrate of the labeling enzyme ALP When aminophenyl phosphate (pAPP) is supplied to the analysis system from the upstream of the sensing unit, it is converted to p-aminophenol (pAP) (Reaction Formula 1). At this time, silver ions (Ag ⁺ , water-soluble) coexist. If it is allowed to stand, silver (Ag, water-insoluble) precipitates (Reaction Formula 2) and precipitates on the sensing part (see FIG. 12). The silver deposited on the sensing part (working electrode) is re-oxidized on the sensing part according to the potential applied to the working electrode. Along with reoxidation of silver, a current flows between the working electrode and the counter electrode (see Reaction Formula 3, FIG. 13). Therefore, by measuring this current, the amount of reoxidized silver can be measured, and the amount of silver deposited can also be measured.

(Biomolecular sensor)
Next, the biomolecule sensor 140 of this embodiment will be described. In addition, about the component of the same aspect as the nucleic acid sensor 40 demonstrated in 2nd Embodiment, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  As shown in FIGS. 14 and 15, the biomolecule sensor 140 includes a base material (electrode substrate) 141, a base material 141, a reference electrode 150 formed on the main surface 141 a of the base material 141, and a working electrode 160. And a counter electrode 170. The reference electrode 150, the working electrode 160, and the counter electrode 170 each extend in a streak shape in the same direction. The working electrode 160 and the counter electrode 170 are disposed across the reference electrode in plan view. Further, the working electrode 160 has an area securing portion 160a that extends in a circular shape at the end in the length direction in order to secure an area as a sensing portion.

  The biomolecule sensor 140 electrochemically analyzes the insoluble substance precipitated on the surface of the sensing unit. The biomolecule sensor 140 captures a change in charge on the surface of the sensing unit (the surface of the working electrode 160) as a change in current. The biomolecule sensor 140 applies a voltage based on the reference electrode 150 to the working electrode 160 on which silver (Ag) is deposited, and reoxidizes the silver deposited on the working electrode 160 into ions (Ag ions). Thus, silver deposition is detected using the electrochemical change at the working electrode 160 as a current change.

(Reference electrode)
The reference electrode 150 includes a base layer 151, an Ag layer 52 containing silver as a main component, an AgCl layer 53 containing silver chloride as a main component, and a SAM film (self-organized) in this order from the main surface 141 a side of the substrate 141. Monomolecular film) 54. In this embodiment, the base layer 151 is a carbon electrode formed by printing a conductive carbon paste. The Ag layer 52, the AgCl layer 53, and the SAM film 54 have the same configuration as that of the second embodiment.

(Working electrode, counter electrode)
The working electrode 160 and the counter electrode 170 are carbon electrodes similar to the base layer 151 of the reference electrode 150, and are printed and formed simultaneously with the base layer 151.

  The biomolecule sensor 140 of this embodiment has a reference electrode 150 in which a SAM film 54 is formed on the surface of an AgCl layer 53. Thereby, the exchange of Cl ions between the AgCl layer 53 and the aqueous solution is suppressed, the elution of Cl ions from the AgCl layer 53 does not easily occur, and the potential drift of the reference electrode 150 can be reduced. It becomes possible to measure the current value between the working electrode 160 and the counter electrode 170 at a stable potential. The biomolecule sensor detects silver deposition and deposits by measuring a current value flowing between the working electrode 160 and the counter electrode 170 when a predetermined voltage is applied to the working electrode 160 with reference to the reference electrode. Measure the amount. For this reason, by reducing the drift of the potential of the reference electrode, it is possible to reduce the variation in the measured value, and it is possible to accurately detect the biomolecule to be measured.

  The biomolecule sensor 140 of this embodiment may be combined with the fluidic device 1 (FIG. 11) of the fourth embodiment. In that case, a biomolecule sensor 140 is provided in place of the nucleic acid sensor 40 of the fluid device 1. Furthermore, in that case, since it is not necessary to disrupt the exosome, the exosome purification unit 2 is omitted.

  Although various embodiments of the present invention have been described above, each configuration in each embodiment and combinations thereof are examples, and addition, omission, replacement, and configuration of configurations are within the scope without departing from the spirit of the present invention. Other changes are possible. Further, the present invention is not limited by the embodiment.

  Next, an example explains the present invention. In addition, this invention is not limited to a following example.

[Production of reference electrode]
A procedure for manufacturing the reference electrode 50 according to the embodiment will be described with reference to FIG. In addition, description about the preparation procedure of the working electrode 60 is abbreviate | omitted here.
First, the substrate 41 is cleaned for 15 minutes with a mixed solution of H ₂ SO ₄ and H ₂ O ₂ (mixing ratio 1: 1, temperature 200 ° C.).
Next, DC sputtering is performed on the substrate 41 using a mask having an opening with a width of 0.9 mm. Thereby, a Cr layer (30 nm or less, not shown in FIG. 1), a gold base layer 51 (150 nm or less), and an Ag layer 52 (3 μm or less) are sequentially formed on the main surface 41a of the substrate 41 made of glass. To do.
Next, an AgCl layer 53 (0.1 nm or less) was formed on the surface of the Ag layer 52 by immersing in an aqueous solution of 1 mol / L FeCl ₃ for 1 minute.
Next, the SAM film 54 containing mercaptohexanol as the SAM film constituent material 54a was formed on the surface of the AgCl layer 53 by being immersed in an aqueous solution of 1 mmol / L mercaptohexanol at room temperature for 24 hours.
The reference electrode 50 of the example was manufactured through the above steps.
In addition, a reference electrode of a comparative example in which the step of forming the SAM film 54 is omitted in the above-described steps was separately prepared.

[Characteristic evaluation of reference electrode]
Next, the reference electrodes of Examples and Comparative Examples were evaluated.
FIG. 16 is a graph showing measurement results of changes in potential difference with time when reference electrodes of Examples and Comparative Examples immersed in a measurement buffer and a commercially available silver-silver chloride electrode are connected to each other. The silver-silver chloride electrode is large in comparison with the reference electrodes of the examples and comparative examples, but has excellent potential stability, and should be used as a standard for measuring the stability of the reference electrodes of the examples and comparative examples. Can do.
As shown in FIG. 16, the change in potential with time of the reference electrode of the example was smaller than that of the reference electrode of the comparative example. The reference electrode of the comparative example had a potential change of −1.4 mV / hr, whereas the reference electrode of the example remained at a potential change within ± 0.2 mV / hr.

FIG. 17 is a graph showing measurement results of temporal changes in potential difference when the reference electrodes of Examples and Comparative Examples immersed in a measurement buffer and the working electrode made of gold are connected to each other.
As shown in FIG. 17, the potential change with time of the reference electrode of the example was smaller than that of the reference electrode of the comparative example. In the reference electrode of the comparative example, Cl ions were eluted from the AgCl layer and the Cl ion concentration in the measurement buffer solution was changed, so that it was considered that there was a significant potential fluctuation. On the other hand, in the reference electrode of the example, since the elution of Cl ions from the AgCl layer is suppressed by the SAM film, there is no fluctuation in the Cl ion concentration of the measurement buffer solution, and the potential is stable. it is conceivable that.

  From the results shown in FIGS. 16 and 17, the reference power of the example having the SAM film on the surface of the AgCl film is superior in potential stability to the reference electrode of the comparative example having no SAM film. Was confirmed.

[Nucleic acid detection test (application to nucleic acid sensor)]
The fluidic device 21 shown in FIG. 10 was configured using the reference electrode produced through the above-described steps, and nucleic acid (miRNA) detection tests at various concentrations were performed by the nucleic acid sensor 40 in the fluidic device 21.

  In this test, miR16 is the target. A capture probe 70 having 100% homology with miRNA was formed on the working electrode 60. Further, the homology between miR21 to be tested as a negative control and the capture probe 70 was 32%. The potential shift of the working electrode relative to the reference electrode 50 was measured for various concentrations of miR21 (target) and miR21 (negative control). The measurement results are shown in FIG. The unit [M] on the horizontal axis in FIG. 18 represents [mol / L].

  From the measurement results, it was confirmed that a potential shift occurred depending on the concentration of the target miR16. It was also confirmed that the potential shift with respect to the non-target miR21 was sufficiently small. In addition, the sensitivity of the potential shift with respect to miR16 was 20 fM.

[Enzyme level measurement test with silver insolubilization reaction (application to immunoassay)]
As an application example involving a chemical reaction that occurs in the presence of silver ions, an enzyme amount measurement test involving a silver insolubilization reaction was performed.
First, the biomolecule sensor 140 shown in FIGS. 14 and 15 was produced by the following procedure. First, a commercially available carbon electrode chip (a round carbon electrode manufactured by Biodevice Technology Co., Ltd.) was prepared. Two of the carbon electrodes on the chip (base material 141) were used as a working electrode 160 and a counter electrode 170, respectively. Mercaptohexanol (hereinafter referred to as MCH) was modified on the surface of the AgCl layer 53 of the reference electrode 150 to form a SAM film 54. The biomolecule sensor 140 was produced through the above steps. This biomolecule sensor is referred to as a “MCH-coated” biomolecule sensor in which a reference electrode is MCH coated.
In addition, as a biomolecule sensor for comparison, a biomolecule sensor produced by the same procedure was prepared except that the reference electrode was not modified with MCH (the SAM film 54 was not formed). The biomolecule sensor for comparison is referred to as a “MCH-coated” biomolecule sensor.

Next, the following tests were performed on the above-described “MCH-coated” and “MCH-coated” biomolecule sensors.
First, as shown in FIG. 19, magnetic fine particles in which ALP (alkaline phosphatase; Wako Pure Chemical Industries, Ltd.) is chemically immobilized are captured on a working electrode by a 2 mm × 2 mm × 4 mm neodymium magnet, and pAPP (abcam) ) And AgNO ₃ (Wako Pure Chemical Industries, Ltd.) (AgNO ₃ ; 0.125 mmol / L, pAPP; 2 mmol / L, MgSO ₄ ; 2 mmol / L, tris (2-amino-2-hydroxymethyl-1) , 3-propanediol); 12.5 mmol / L) for 3 minutes. Thereby, the silver deposition reaction shown by Reaction Formula 1 and Reaction Formula 2 (see the embodiment) was performed. Thereafter, the electrode is cleaned with KNO ₃ solution 1 mol / L, again using a 1 mol / L of KNO ₃ was added electrochemical analyzer als1232B (ALS Co.), the differential pulse voltammetry (DPV) measurement, Scheme A silver reionization reaction represented by 3 (see the embodiment) was performed, and the change in current value was measured. Furthermore, it was brought into contact with a buffer solution containing silver ions (AgNO ₃ ; 2 mmol / L, MgSO ₄ ; 2 mmol / L, Tris; 12.5 mmol / L) for 1 hour, and before and after that, on the working electrode again as described above. Silver deposition and reionization were carried out to determine the peak potential in the DPV measurement.

  As shown in FIG. 20, in the biomolecule sensor “without MCH coating”, when the reference electrode was brought into contact with a buffer solution containing silver ions for 60 minutes, the peak potential was shifted to the negative side by about 9%. On the other hand, in the biomolecule sensor with “MCH coating”, since the shift amount of the peak potential is suppressed to 3%, the SAM film formation causes the reference electrode to occur during contact with the buffer containing silver ions. It was confirmed that the potential shift can be suppressed.

DESCRIPTION OF SYMBOLS 1, 21 ... Fluid device, 2 ... Exosome refinement | purification part, 3 ... Biomolecule refinement | purification part (object purification part), 4 ... Biomolecule detection part 5, 6, 10, 11, 12, 33 ... Flow path, 7, 8, 9 ... 3rd waste liquid tank, 24 ... Insulating film, 25 ... Circuit board, 25a ... Control circuit, 30 ... Channel base material, 40 ... (Nucleic acid) sensor, 41, 141 ... Substrate (electrode board), 50 Reference electrode (reference electrode), 51, 61, 151 ... Base layer, 52 ... Ag layer (first member), 53 ... AgCl layer (second member), 54, 62 ... SAM film (self-organized) SAM membrane constituent material, 60, 160 ... Working electrode (working electrode), 70, 70A, 70B ... Capture probe, 75 ... Capture part, 102 ... Antibody, 103 ... Antigen, 104 ... ALP label Antibody, 105 ... magnetic fine particle, 140 ... (biomolecule) Sir, 170 ... counter electrode (counter electrode)

Claims (18)

A first member comprising silver as a main component;
A second member mainly composed of silver chloride and in contact with at least a part of the first member;
An electrode comprising: a self-assembled monolayer formed on the surface of the second member.   The electrode according to claim 1, wherein the self-assembled monolayer includes an alkanethiol derivative chemically adsorbed on a surface of the second member.   The electrode according to claim 1, wherein the second member is a layer formed on a surface of the first member.   The electrode according to claim 1, wherein the electrode is formed on a substrate, and the first member is a layer formed on a surface of the substrate. A first member mainly composed of silver, a second member mainly composed of silver chloride and in contact with at least a part of the first member, and a self-organized unit formed on the surface of the second member. A reference electrode having a molecular film;
A sensor comprising: a working electrode having a detection unit that detects an object contained in a solution to be measured. The object is a biomolecule;
The said working electrode has the capture part which can react with the said biomolecule on the surface, and detects the said biomolecule by measuring the electrical potential change accompanying reaction of the said biomolecule in the said capture part. Sensor. The biomolecule is a nucleic acid;
The capture unit has a capture probe having a sequence capable of hybridizing with the nucleic acid to be detected, and detects the nucleic acid by measuring a potential change caused by hybridization between the capture probe and the nucleic acid. 6 sensors.   The sensor according to claim 7, wherein the capture probe has a first part composed of a sequence capable of hybridizing with the nucleic acid to be detected, and a stem part forming a double strand.   The sensor according to any one of claims 5 to 8, wherein a self-assembled monolayer having the same composition as the reference electrode is formed on the surface of the working electrode.   The sensor according to claim 7 or 8, wherein the nucleic acid to be detected is miRNA. The object is a biomolecule;
The biomolecule and a specific substance having a selective interaction with the biomolecule are reacted, and a soluble substance is reacted to correlate with the abundance of the biomolecule contained in the solution to be measured to form an insoluble substance. The sensor according to claim 5, wherein the sensor is converted and deposited on the working electrode, and the deposited insoluble substance is electrically detected.   The sensor according to claim 11, further comprising a counter electrode, and measuring a deposition amount of the insoluble substance by measuring a current value when a potential is applied between the working electrode and the counter electrode.   The sensor according to claim 11 or 12, wherein the soluble substance is a metal ion.   A fluid device comprising the sensor according to claim 5.   The fluid device according to claim 14, further comprising an object purification unit that extracts the object in the sample. A circulation channel for circulating the liquid containing the object,
The fluid device according to claim 14 or 15, wherein the sensor is provided in the circulation channel. An electrode substrate on which the electrode according to any one of claims 1 to 4 is formed;
A flow path base material laminated on the electrode substrate so that a flow path through which a liquid to be measured flows is formed, and the electrode is disposed in the flow path;
And a circuit board having a control circuit for controlling the electrodes of the electrode board. Forming a layered first member mainly composed of silver on one surface side of the substrate;
Forming a layered second member mainly composed of silver chloride on the surface of the first member;
Forming a self-assembled monolayer on the surface of the second member.