WO2009133679A1 - Biosensor electrode manufacturing method and biosensor manufacturing method - Google Patents

Abstract

A step for manufacturing a carbon electrode includes: a substep for adding nanocarbon to a solvent containing no binder agent so as to prepare a dispersed liquid (carbon nanotubes dispersion liquid); a substep for applying the nanocarbon-dispersed liquid to a substrate (12) so as to form a nanocarbon layer; and a substep for solidifying an antibody or a catalyst to the nanocarbon layer.

Description

Biosensor electrode manufacturing method and biosensor manufacturing method

The present invention relates to a biosensor electrode manufacturing method and a biosensor manufacturing method.

Conventionally, nanocarbons such as carbon nanotubes (CNT) have been used for carbon electrodes. Nanocarbon is expected in terms of increasing the sensitivity of a biosensor because it has excellent electrical conductivity and promotes an electron transfer reaction with a biomolecule derived from the nanostructure.
Such a carbon electrode is manufactured by the following manufacturing method.
Patent Document 1 discloses a method in which a carbon nanotube is micelleed with a mixture of a surfactant and a polymer electrolyte solution, dispersed in an aqueous solvent, and then the micelle is decomposed to form a thin film of the carbon nanotube. It is disclosed.
Patent Document 2 discloses that carbon nanotubes are dispersed in an optical modeling resin, and the optical modeling resin is cured by light irradiation to form an electrode.
Further, Patent Document 3 discloses a method of forming a carbon nanotube film by a dry method. Specifically, a raw material gas is sprayed on the catalyst supporting surface of the substrate to grow carbon nanotubes on the catalyst supporting surface.

JP 2007-136645 A JP 2006-260938 A JP 2007-126318 A

In recent years, improvement in the performance of biosensors (for example, improvement in measurement accuracy) has been demanded. However, when electrodes manufactured by the methods disclosed in Patent Documents 1 to 3 are used, such demands can be met. difficult.
This is considered to be due to the following reasons.
In the production methods disclosed in Patent Documents 1 and 2, carbon nanotubes are dispersed in a polymer material such as a resin. This polymer material serves as a binder material for immobilizing the carbon nanotubes. However, since a polymer material is inserted between the carbon nanotubes, the resistance value between the carbon nanotubes is increased. As a result, the sensitivity of the biosensor decreases.
On the other hand, in the manufacturing method disclosed in Patent Document 3, although a polymer material is not used, the catalyst supported on the substrate adversely affects the sensitivity of the biosensor.

The present invention provides a method for producing a biosensor electrode and a method for producing a biosensor that can improve the performance of the biosensor.

According to the present invention, a step of creating a liquid in which nanocarbon is added to a solvent not containing a binder agent, and a step of applying the liquid containing the nanocarbon on a substrate to form a nanocarbon layer. And a method for producing an electrode for a biosensor comprising the step of immobilizing an antibody or an enzyme on the nanocarbon layer.
Here, nanocarbon is any of carbon nanotubes, carbon nanohorns, carbon nanocones, carbon nanofilaments, and fullerenes.
Further, according to the present invention, there is provided a biosensor manufacturing method including the above-described biosensor electrode manufacturing method, wherein an alignment mark is formed at a position below the nanocarbon layer of the substrate. A step of mounting the biosensor electrode on a mounting substrate, and the step of mounting the biosensor electrode on the mounting substrate includes forming an alignment mark on the biosensor electrode substrate on the mounting substrate. A biosensor manufacturing method can be provided in which alignment is performed using the alignment mark and the biosensor electrode is mounted on the mounting substrate.

ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the electrode for biosensors and the manufacturing method of a biosensor which can improve the performance of a biosensor are provided.

The above-described object and other objects, features, and advantages will be further clarified by a preferred embodiment described below and the following drawings attached thereto.

It is sectional drawing which shows the biosensor concerning one Embodiment of this invention. It is a figure which shows typically the state of a carbon nanotube and an antibody. It is a top view which shows a biosensor main body and a mounting substrate.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The present embodiment will be described with reference to FIGS.
FIG. 1 discloses a biosensor 1 of the present embodiment.
The biosensor 1 includes a biosensor body 11 including a substrate 12, a working electrode 13 formed on the substrate 12, a counter electrode 14, and a reference electrode 15, and a mounting substrate 16.
The substrate 12 and the working electrode 13 constitute a carbon electrode for the biosensor of the present invention.

The substrate 12 is an insulating substrate and is a light transmissive substrate. The substrate 12 is glass, plastic, or the like, and the material is not particularly limited, but glass having high strength and high light transmission is preferable. In the present embodiment, light-transmitting means transmitting visible light, and it is only necessary to transmit light through the light-transmitting object to such an extent that the opposite side can be visually recognized by the naked eye. Specifically, the light transmittance of visible light is preferably 70% or more.

The working electrode 13 includes a carbon nanotube thin film (nanocarbon layer) 131 formed on the substrate 12, a layer 132 containing an antibody and an enzyme provided so as to cover the thin film 131, and a thin film 131 provided on the layer 132. And a polymer film 134 provided so as to cover the surface.

The thin film 131 is formed by depositing carbon nanotubes. As shown in FIG. 2, the carbon nanotubes intersect each other so as to be in direct contact with each other. In FIG. 2, reference numeral 131A indicates a carbon nanotube, and a dotted line A indicates an intersection of the carbon nanotubes 131A.
Since the polymer film 134 is provided on the thin film 131, a polymer may be interposed between some of the carbon nanotubes 131A (less than 1% of all the carbon nanotubes). The carbon nanotubes 131A (99% or more of all carbon nanotubes) are in direct contact with each other.
Further, the carbon nanotube 131A is in direct contact with the substrate 12, and the thin film 131 is physically fixed (adsorbed) to the substrate 12 (see dotted line B in FIG. 2).
Since the polymer film 134 is provided on the thin film 131, a polymer may be interposed between some of the carbon nanotubes 131A and the substrate 12, but most of the carbon nanotubes 131A are in direct contact with the substrate 12. is doing.

Various types of carbon nanotubes can be used. For example, they are produced by a laser ablation method, a CoMoCAT method (a method of the University of Oklahoma, USA), a direct-injection pyrolysis synthesis method, or a high pressure carbon monoxide process method. Kinds are preferably used. The laser ablation method is more preferable from the viewpoint of cost.
The thin film 131 is light-transmitting, and it is sufficient that the thin film 131 has such a transparency that the substrate 12 can be visually recognized through the thin film 131. Specifically, the light transmittance of visible light is preferably 70% or more.

Carbon nanotubes having a diameter of 0.8 to 1.3 nm and a length of several μm are preferably used from the viewpoints of quality, difficulty of peeling from the substrate 12, and the like.

The thickness of the thin film 131 is preferably 10 nm or more and 1 μm or less. More preferably, it is 20 nm or more and 50 nm or less. If the thickness of the thin film 131 is too thin, the electrical conductivity decreases, which is not preferable from the viewpoint of reducing the amount of antibody or enzyme immobilized. On the other hand, if it is too thick, the light transmittance is lowered, which is not preferable.

The layer 132 containing an antibody and an enzyme shown in FIG. 1 is obtained by fixing an antibody and an enzyme on a thin film 131.
In the antibody immobilization method, the carbon nanotube thin film 131 is preliminarily treated with an immobilized protein, and then a solution containing the antibody is brought into contact therewith. For example, as shown in FIG. 2, the antibody 132A is immobilized on the carbon nanotube 131A via the immobilized protein 132C.
The immobilized protein has a function for immobilizing the antibody on the carbon nanotube.
As specific immobilized proteins, proteins containing electrochemically active amino acids such as tryptophan and tyrosine may be used, and streptavidin, protein A, protein G and the like are preferably used. Needless to say, if the above-mentioned electrochemically active amino acid is contained in the measurement target substance, these amino acids may not be contained in the immobilized protein. Moreover, even if it is not contained in both the substance to be measured and the immobilized protein, the solution used at the time of measurement may contain an electrochemically active amino acid. These selections can be appropriately changed depending on the substance to be measured. As an antibody to be used, it is possible to apply a wide range of antigens, such as human chorionic gonadotropin antibody and trinitrotoluene antibody, from an antigen having a large molecular weight to a small antigen.

As an enzyme immobilization method, a method using a crosslinking reaction between albumin and glutaraldehyde is preferably used because it can be produced at low cost. As an enzyme to be used, an oxidase such as glucose oxidase, lactate oxidase, or urate oxidase is mainly used, but a dehydrogenase can also be used using a mediator or the like.
The layer 132 is a layer containing an antibody and an enzyme, but may be a layer having only one of an antibody and an enzyme.

As shown in FIGS. 1 and 2, the polymer film 134 covers the layer 132 and the thin film 131 from the upper surface side, and is provided to firmly fix the thin film 131 to the substrate 12. The polymer film 134 also serves to reinforce that the antibody or the like is immobilized on the thin film 131.
Examples of the polymer film 134 include polyvinyl alcohol, polyacetylene, polythiophene, proteins such as albumin and gelatin, and the like.
Of these, polyvinyl alcohol is preferably used because it is inexpensive and the solution can be easily adjusted.

The counter electrode 14 is disposed so as to face the working electrode 13 and includes, for example, platinum. The reference electrode 15 includes, for example, silver. Both the counter electrode 14 and the reference electrode 15 are formed on the substrate 12.

As shown in FIG. 1, the biosensor body 11 having the above configuration is mounted on a mounting substrate 16.
The mounting substrate 16 is an insulating substrate, for example, a flexible substrate. The mounting substrate 16 is preferably made of a material excellent in water resistance, heat resistance, and chemical resistance, and plastics that can be manufactured at low cost are preferable.

A wiring (not shown) is formed on the mounting substrate 16, and the biosensor body 11 is connected to the wiring via a bonding wire W. Sealing agent E is provided on the bonding wire portion.

Platinum, gold, and aluminum are appropriately selected as the material of the bonding wire W, and inexpensive aluminum is preferably used.
The sealant E is not particularly limited as long as it uses a material that waterproofs the bonding wire W and further the connection portion between the bonding wire W and the biosensor main body 11 and protects it from breakage. A silicone resin is preferably used from the viewpoint of reliability.
The biosensor body 11 is connected to the electrochemical measurement device via the mounting substrate 16.

Next, the manufacturing method of the above biosensor 1 is demonstrated.
The manufacturing method of the biosensor 1 of the present embodiment includes a step of manufacturing the biosensor main body 11 and a step of mounting the biosensor main body 11 on the mounting substrate 16.
The manufacturing method of the biosensor body 11 includes a step of manufacturing a carbon electrode including the working electrode 13 and the substrate 12, and a step of manufacturing the counter electrode 14 and the reference electrode 15.

First, an outline of a process for manufacturing a carbon electrode will be described.
The steps of manufacturing the carbon electrode include adding a nanocarbon (carbon nanotube) to a solvent that does not contain a binder agent to create a dispersed liquid (carbon nanotube dispersion), and the above-mentioned liquid in which nanocarbon is dispersed. And a step of coating on the substrate 12 to form a nanocarbon layer (thin film 131), and a step of immobilizing an antibody or an enzyme on the nanocarbon layer.
Here, the binder agent is for adhering nanocarbons to each other, and is, for example, a polymer material such as a polymer resin.

Next, the process for manufacturing the carbon electrode will be described in detail.
First, carbon nanotubes are prepared, and the carbon nanotubes are washed using an acid or alkali solution. Specifically, carbon nanotubes are dispersed in an acid or alkali solution and washed.
The acid solution preferably has a pH of 4 or less, and examples thereof include nitric acid and hydrochloric acid. The concentration of nitric acid, hydrochloric acid, etc. in the acid solution is preferably 0.01M or more and 1M or less. As an alkaline solution, it is preferable that pH is 8 or more, for example, ammonia, sodium hydroxide, potassium hydroxide etc. are mentioned. The concentration is preferably 0.01M or more and 3M or less.
In the carbon nanotube production process, the used catalyst may be slightly attached to the carbon nanotube. Such a catalyst may affect the sensitivity of the biosensor.
Therefore, by providing this step, the catalyst adhering to the carbon nanotube is removed, and the sensitivity of the biosensor can be prevented from being lowered.
Whether or not the catalyst has been removed can be confirmed by, for example, EDX (energy dispersive X-ray spectrometry).

Next, the carbon nanotubes are dispersed in a solvent that does not contain a binder agent for bonding the carbon nanotubes together to create a carbon nanotube dispersion.
As the solvent, an organic solvent can be used. As the organic solvent, dichloroethane, dimethylsulfone, acetone, ether, DMF (dimethylformamide) and the like are preferable. Of these, it is preferable to use dichloroethane having good dispersibility of carbon nanotubes.
As a method of dispersing the carbon nanotubes in the solvent, the solvent may be stirred with a stirrer. However, if the method of dispersing by treating with ultrasonic waves (stirring by ultrasonic waves) is used, the carbon nanotubes are efficiently dissolved in the solvent. Can be dispersed. Among these, carbon nanotubes can be more effectively dispersed in the solvent by using dichloroethane and further stirring with ultrasonic waves.
Here, the carbon nanotube dispersion liquid is composed only of carbon nanotubes and a solvent, and does not contain a binder agent, a surfactant or the like.

Next, a solvent in which nanocarbon is dispersed (carbon nanotube dispersion) is applied on the substrate 12 to form a carbon nanotube thin film 131. A spin coating method or a dip coating method is preferably used as the coating method, but the dip coating method is preferable from the viewpoint that the thickness of the thin film 131 can be increased efficiently. Even when the spin coating method is applied, it is possible to increase the thickness of the carbon nanotube thin film 131 by increasing the number of coatings as in the case of the dip coating method.

Thereafter, the solvent is evaporated, and the enzyme and the antibody are immobilized on the carbon nanotube thin film 131 to form the layer 132.
Enzyme and antibody immobilization methods are as described above.

Next, a polymer film 134 is formed.
Specifically, a solution containing a polymer (for example, a polyvinyl alcohol solution) is applied on the layer 132.
Thereafter, the counter electrode 14 and the reference electrode 15 are formed on the substrate 12.
The biosensor body 11 is manufactured through the above steps.

Next, with reference to FIG. 3, the process of mounting the biosensor body 11 on the mounting substrate 16 will be described.
First, an alignment mark is formed on the substrate 12 of the biosensor body 11 in advance. As a method of forming the alignment mark M1, a metal such as gold or silver may be formed in advance by sputtering or the like and patterned, or may be scratched with a glass cutter or the like. The former is preferable when the size of the substrate 12 is small, and the latter is preferable when the size of the substrate 12 is large.
Note that the step of forming the alignment mark M1 is performed on the substrate 12 before the electrode is formed. In the state where the electrode is formed, the alignment mark M1 is covered with the carbon nanotube thin film 131 of the working electrode 13 or the like.

On the other hand, the alignment mark M2 is also formed on the mounting substrate 16. When the wiring is formed on the mounting substrate 16, it is preferable to pattern the alignment mark with the same material as the wiring, but the alignment mark M2 is not limited to this.

Next, the alignment mark M2 formed on the surface of the mounting substrate 16 is aligned with the alignment mark M1 formed on the substrate 12, and the biosensor body 11 is placed on the mounting substrate 16.
Here, since the substrate 12 is light transmissive, and the carbon nanotube thin film 131 and the polymer film 134 are also light transmissive, in this embodiment, alignment can be easily performed. .
Thereafter, the mounting substrate 16 and the biosensor main body 11 are electrically connected using the bonding wire W, and the connection portion is sealed with a sealant.

Next, the effect of this embodiment is demonstrated.
In the conventional method for producing a carbon electrode, carbon nanotubes are dispersed in a polymer material such as a resin, and this polymer material is applied onto a substrate. Resistance value becomes high.
On the other hand, in the present embodiment, the thin film 131 is formed by dispersing the carbon nanotubes in a solvent that does not contain the binder agent and applying the solvent onto the substrate 12.
Therefore, the carbon nanotubes in the thin film 131 are easily brought into direct contact with each other, and it is possible to prevent the resistance value between the carbon nanotubes from increasing. Thereby, the performance of the biosensor using the carbon electrode of this embodiment can be improved.

In the conventional carbon electrode manufacturing method, a catalyst is supported on a substrate to form a carbon nanotube thin film. In this method, iron, cobalt, nickel or the like is generally used as a catalyst, but such a catalyst reduces the sensitivity of the biosensor. This is because the catalyst generates an interference current in current-potential measurement or constant potential measurement (for example, Fe → Fe2 ++ 2e−). In such a carbon electrode, a catalyst is supported on a substrate to form a carbon nanotube thin film. After forming a carbon nanotube thin film, even if an attempt is made to remove the catalyst with an acid or alkali solution, the carbon nanotube This thin film makes it difficult for an acid or alkali solution to penetrate to the substrate surface. Therefore, it is difficult to remove the catalyst.
On the other hand, in this embodiment, the thin film 131 is formed by applying a solvent in which carbon nanotubes are dispersed on the substrate 12, and the catalyst is supported on the substrate as in the conventional case, and the carbon nanotube thin film is formed. Does not form. Therefore, the performance of the biosensor can be improved.
In particular, in the present embodiment, the carbon nanotubes are washed with an acid or alkali solution, and the catalyst adhered in the carbon nanotube production process is removed, so that the catalyst can be reliably removed, and the performance of the biosensor is further improved. Can be improved.

Furthermore, in this embodiment, the polymer film 134 is formed so as to cover the thin film 131 of carbon nanotubes. The polymer film 134 can prevent the thin film 131 from peeling off the substrate 12. Since the polymer film 134 is applied on the thin film 131 after the thin film 131 is formed, the polymer hardly enters between the carbon nanotubes.

In this embodiment, since the carbon nanotube thin film 131 is formed by a wet method, the thin film 131 is light transmissive. In the present embodiment, the substrate 12 is also light transmissive. Therefore, the position of the biosensor body 11 with respect to the mounting substrate 16 is accurately determined by using the alignment mark M1 formed on the portion below the thin film 131 of the substrate 12 and the alignment mark M2 formed on the mounting substrate 16. Can be adapted.

It should be noted that the present invention is not limited to the above-described embodiments, and modifications, improvements, and the like within the scope that can achieve the object of the present invention are included in the present invention.
For example, in the above embodiment, one working electrode 13, one counter electrode 14, and one reference electrode 15 are formed on one substrate 12. However, the present invention is not limited to this, and a large-sized substrate is prepared, and the working electrode, the counter electrode, After forming many sets of reference electrodes, the biosensor body 11 may be manufactured by cutting the substrate.
Furthermore, in the above embodiment, the carbon nanotubes are washed with an acid or alkali solution to remove the catalyst, but this step may be omitted. By doing in this way, the manufacturing process of a biosensor can be simplified.

Moreover, in the said embodiment, although the carbon nanotube was used for the thin film 131 which comprises a carbon electrode, the carbon used for a carbon electrode should just be nanocarbon, carbon nanohorn, carbon nanocone, carbon nanofilament, It may be fullerene.
This application claims priority based on Japanese Patent Application No. 2008-118483 filed on Apr. 30, 2008, the entire disclosure of which is incorporated herein.

Next, examples of the present invention will be described.
Example 1
A 10 × 10 × 0.7 mm glass substrate (light-transmitting substrate) was prepared, and ultrasonic cleaning was performed in a nitric acid-hydrogen peroxide solution for 5 minutes. Subsequently, a diamond-shaped mark was formed at the center of the glass substrate as an alignment mark by sputtering at two locations. The material of the alignment mark is platinum, and the size of the alignment mark is 200 μm square.
Subsequently, CoMoCAT carbon nanotubes were dispersed in 1,2-dichloroethane. In dispersion, ultrasonic treatment was performed for 2 hours to prepare a solution having a carbon nanotube concentration of 10 ppm. The carbon nanotubes have a diameter of 0.8 to 1.6 nm and a length of several μm.
The solution in which the carbon nanotubes are dispersed does not include a binder agent or a surfactant, and includes carbon nanotubes and a solvent.
Next, the glass substrate was immersed in the solution and pulled up with a dip coater (pickup speed: 0.2 mm / sec) to coat the carbon nanotubes on the substrate surface. This pulling operation was performed 10 times and 20 times, and a carbon electrode of a carbon nanotube thin film having a thickness of about 10 nm was manufactured.
Next, a 22.5 w / v% albumin solution containing glucose oxidase and 1 v / v% glutaraldehyde was spin-coated at 3000 rpm to immobilize the enzyme on the carbon nanotube thin film. Here, although the alignment mark formed on the glass substrate was covered with the carbon nanotube thin film, it could be visually confirmed, and the carbon nanotube thin film was light-transmitting.
Moreover, a reference electrode and a platinum counter electrode were formed on the substrate, and a glucose biosensor main body was manufactured.
Thereafter, a printed board (mounting board) made of polyimide resin of 15 × 100 × 0.2 mm in which two rhombus alignment marks were formed with copper was prepared.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the biosensor thus produced, glucose in the solution (glucose concentration 1 mM) was measured by the three-electrode method.

(Example 2)
A 10 × 10 × 0.7 mm glass substrate (light-transmitting substrate) was prepared, and ultrasonic cleaning was performed in a nitric acid-hydrogen peroxide solution for 5 minutes. Subsequently, a diamond-shaped mark was formed at the center of the glass substrate as an alignment mark by sputtering at two locations. The material is platinum and the size is 200 μm square.
Subsequently, laser ablation carbon nanotubes were dispersed in 1,2-dichloroethane. As a dispersion method, ultrasonic treatment was performed for 2 hours. And the solution whose density | concentration of a carbon nanotube is 10 ppm was created. The carbon nanotube has a diameter of about 1.3 nm and a length of several μm. The solution in which the carbon nanotubes are dispersed does not include a binder agent or a surfactant, and includes carbon nanotubes and a solvent.
Subsequently, the glass substrate was dipped in the solution and pulled up with a dip coater (pickup speed: 0.2 mm / sec) to coat the carbon nanotubes on the substrate surface. This pulling operation was performed 20 times, and a carbon electrode of a carbon nanotube thin film having a thickness of about 16 nm was manufactured.
Next, the carbon electrode was immersed in a solution of 1 mg / 0.2 ml of human chorionic gonadotropin antibody (mouse immunized monoclonal antibody manufactured by Hitest) for 1 hour. Then, it is immersed in 3 mM 1,3-diaminobenzene (Aldrich Co., USA, containing phosphate buffer solution of pH 7.4 and 0.1 M sodium chloride), and 0 to 0.8 V is 2 mV. Swept 100 times at / s and applied for 5 hours at 0.65 V to immobilize the antibody described above. Subsequently, it was immersed in 1 w / v% polyvinyl alcohol for 1 hour, and the surface of the antibody was coated with polyvinyl alcohol (the carbon nanotube thin film would be coated with polyvinyl alcohol through the antibody layer). Here, the alignment mark formed on the glass substrate was covered with a carbon nanotube thin film, an antibody layer, and a polyvinyl alcohol layer, but it can be visually confirmed, and the carbon nanotube thin film is light-transmitting. I understand.
Thereafter, a printed board (mounting board) made of polyimide resin of 15 × 100 × 0.2 mm in which two rhombus alignment marks were formed with copper was prepared.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the carbon electrode thus produced as a working electrode, the chorionic gonadotropin concentration (30 nM) in the solution was measured by a square wave voltammetry method using a glass reference electrode and a platinum counter electrode. The measurement conditions are 0.1-1.2 V sweep range, 40 mV pulse potential, 4 Hz frequency, 10 mV step potential.

(Example 3)
A 10 × 10 × 0.7 mm glass substrate (light-transmitting substrate) was prepared, and ultrasonic cleaning was performed in a nitric acid-hydrogen peroxide solution for 5 minutes. Subsequently, a diamond-shaped mark was formed at the center of the glass substrate as an alignment mark by sputtering at two locations. The material of the alignment mark is platinum, and the size of the alignment mark is 200 μm square.
Subsequently, CoMoCAT carbon nanotubes were placed in a 1 M hydrochloric acid solution and subjected to ultrasonic cleaning treatment for 1 hour. And the carbon nanotube was scooped up with the glass filter, the carbon nanotube was rinsed fully with the pure water, and hydrochloric acid was removed completely. Thereby, the catalyst adhering to the carbon nanotube was removed.
Subsequently, the above-mentioned carbon nanotubes were put in 1,2-dichloroethane and sufficiently dispersed by ultrasonic treatment for 2 hours to prepare a solution having a carbon nanotube concentration of 10 ppm. The carbon nanotubes have a diameter of 0.8 to 1.6 nm and a length of several μm. The solution in which the carbon nanotubes are dispersed does not include a binder agent or a surfactant, and includes carbon nanotubes and a solvent.
Next, the glass substrate was immersed in the solution and pulled up with a dip coater (pickup speed: 0.2 mm / sec) to coat the carbon nanotubes on the substrate surface. This pulling operation was performed 10 times and 20 times, and a carbon electrode of a carbon nanotube thin film having a thickness of about 10 nm was manufactured.
Next, a 22.5 w / v% albumin solution containing glucose oxidase and 1 v / v% glutaraldehyde was spin-coated at 3000 rpm to immobilize the enzyme on the carbon nanotube thin film. Here, although the alignment mark formed on the glass substrate was covered with the carbon nanotube thin film, it could be visually confirmed, and the carbon nanotube thin film was light-transmitting.
Moreover, a reference electrode and a platinum counter electrode were formed on the substrate, and a glucose biosensor main body was manufactured.
Thereafter, a printed board (mounting board) made of polyimide resin of 15 × 100 × 0.2 mm in which two rhombus alignment marks were formed with copper was prepared.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the biosensor thus produced, glucose in the solution (glucose concentration 1 mM) was measured by the three-electrode method.

Example 4
A 10 × 10 × 0.7 mm glass substrate (light-transmitting substrate) was prepared, and ultrasonic cleaning was performed in a nitric acid-hydrogen peroxide solution for 5 minutes. Subsequently, a diamond-shaped mark was formed at the center of the glass substrate as an alignment mark by sputtering at two locations. The material of the alignment mark is platinum, and the size of the alignment mark is 200 μm square.
Subsequently, CoMoCAT carbon nanotubes were placed in a 0.5 M sodium hydroxide solution and subjected to ultrasonic cleaning treatment for 1 hour. And the carbon nanotube was scooped up with the glass filter, the carbon nanotube was rinsed fully with the pure water, and the sodium hydroxide solution was removed completely. Thereby, the catalyst adhering to the carbon nanotube was removed.
Subsequently, the above-mentioned carbon nanotubes were put in 1,2-dichloroethane and sufficiently dispersed by ultrasonic treatment for 2 hours to prepare a solution having a carbon nanotube concentration of 10 ppm. The carbon nanotubes have a diameter of 0.8 to 1.6 nm and a length of several μm. The solution in which the carbon nanotubes are dispersed does not include a binder agent or a surfactant, and includes carbon nanotubes and a solvent.
Next, the glass substrate was immersed in the solution and pulled up with a dip coater (pickup speed: 0.2 mm / sec) to coat the carbon nanotubes on the substrate surface. This pulling operation was performed 10 times and 20 times, and a carbon electrode of a carbon nanotube thin film having a thickness of about 10 nm was manufactured.
Next, a 22.5 w / v% albumin solution containing glucose oxidase and 1 v / v% glutaraldehyde was spin-coated at 3000 rpm to immobilize the enzyme on the carbon nanotube thin film. Here, although the alignment mark formed on the glass substrate was covered with the carbon nanotube thin film, it could be visually confirmed, and the carbon nanotube thin film was light-transmitting.
Moreover, a reference electrode and a platinum counter electrode were formed on the substrate, and a glucose biosensor main body was manufactured.
Thereafter, a printed board (mounting board) made of polyimide resin of 15 × 100 × 0.2 mm in which two rhombus alignment marks were formed with copper was prepared.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the biosensor thus produced, glucose in the solution (glucose concentration 1 mM) was measured by the three-electrode method.

(Comparative Example 1)
A glass substrate of 10 × 10 × 0.7 mm was prepared and subjected to ultrasonic cleaning for 5 minutes in a nitric acid-hydrogen peroxide solution. Subsequently, a diamond-shaped mark was formed at the center of the glass substrate as an alignment mark by sputtering at two locations. The material of the alignment mark is platinum, and the size of the alignment mark is 200 μm square.
Subsequently, CoMoCAT carbon nanotubes were dispersed in 1,2-dichloroethane, and further dispersed by ultrasonic treatment for 2 hours. Subsequently, carbon nanotubes were placed in a 5 w / v% Nafion (registered trademark) solution manufactured by DuPont, which is a polymer electrolyte solution, and further subjected to ultrasonic cleaning treatment for 2 hours, and Nafion (registered trademark) having a concentration of 10 ppm. -A carbon nanotube solution was prepared. The carbon nanotubes have a diameter of 0.8 to 1.6 nm and a length of several μm.
Next, the glass substrate was immersed in the solution and pulled up by a dip coater (pickup speed: 0.2 mm / sec) to coat Nafion (registered trademark) -carbon nanotubes on the substrate surface. This pulling operation was performed 10 times and 20 times, and a carbon electrode of a thin film of Nafion (registered trademark) -carbon nanotubes having a thickness of about 1 μm was manufactured.
Next, a 22.5 w / v% albumin solution containing glucose oxidase and 1 v / v% glutaraldehyde was spin-coated at 3000 rpm, and the enzyme was immobilized on the Nafion (registered trademark) -carbon nanotube thin film. .
Moreover, a reference electrode and a platinum counter electrode were formed on the substrate, and a glucose biosensor main body was manufactured.
Thereafter, a printed board (mounting board) made of polyimide resin of 15 × 100 × 0.2 mm in which two rhombus alignment marks were formed with copper was prepared.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the biosensor thus produced, glucose in the solution (glucose concentration 1 mM) was measured by the three-electrode method.

(Comparative Example 2)
A glass substrate of 10 × 10 × 0.7 mm was prepared and subjected to ultrasonic cleaning for 5 minutes in a nitric acid-hydrogen peroxide solution. Subsequently, a synthetic catalyst composed of fine particles of iron / cobalt was formed on the substrate surface by sputtering.
Subsequently, ethanol was sprayed on the substrate surface as a carbon nanotube source gas to grow carbon nanotubes. The reaction conditions at this time are 850 ° C. for the carbon nanotube growth atmosphere, 50 ° C. for the source gas, a spray rate of 500 ml / min, and a reaction time of 10 seconds. Then, a carbon electrode having a thin film of 1 μm carbon nanotubes was manufactured.
Next, a 22.5 w / v% albumin solution containing glucose oxidase and 1 v / v% glutaraldehyde was spin-coated at 3000 rpm to immobilize the enzyme on the carbon nanotube thin film.
Moreover, a reference electrode and a platinum counter electrode were formed on the substrate, and a glucose biosensor main body was manufactured.
Subsequently, the side surface of the substrate was aligned with the alignment mark of the polyimide resin described above, and the substrate was mounted on the polyimide resin.
Next, the biosensor main body was mounted on a printed circuit board using an actual microscope so that the marks of both the alignments overlapped. Subsequently, the mounting substrate and the biosensor main body were connected by wire bonding, and the connection portion and the entire wire bonding were sealed with silicone resin.
Using the biosensor thus produced, glucose in the solution (glucose concentration 1 mM) was measured by the three-electrode method.

(result)
The results from Example 1 to Comparative Example 2 are shown in Table 1.
The items evaluated were as follows, and each item was evaluated on a 5-point scale.
(1) Base current value indicating the background current output value When the base current value exceeds 1 μA When the one-point base current value exceeds 500 nA and below 1 μA When the two-point base current value exceeds 200 nA and below 500 nA When the 3-point base current value exceeds 50 nA and is 200 nA or less 4-point base current value is 50 nA or less 5 points (2) Indicates the current output value against noise when measuring 1 mM glucose or 30 nM chorionic gonadotropin Signal / noise average value Signal / noise average value is less than 1 1 point Signal / noise average value is 1 or more and less than 2 2 points Signal / noise average value is 2 or more and less than 5 3 Point When the average value of signal / noise is 5 or more and less than 20 4 points When the average value of signal / noise is 20 or more Total 5 points (3) Measurement accuracy (CV (%)) showing variation when each solution of 1 mM glucose or 30 nM chorionic gonadotropin is measured 10 times.
However, for the measurement of chorionic gonadotropin, the operation of immersing the sensor in a 10 mM glycine solution being stirred with a stirrer for 30 seconds and then performing the measurement again was repeated 10 times.
The measurement accuracy (CV (%)) is calculated as follows.
C. V. (%) = (Standard deviation of current value / average value of current value) × 100
When the measurement accuracy exceeds 15% 1 point When the measurement accuracy is 10% or more and 15% or less 2 points When the measurement accuracy is 6% or more and less than 10% 3 points When the measurement accuracy is 3% or more and less than 6% 4 points Measurement accuracy is less than 3% 5 points

From Table 1, the superiority of the sensor according to the present invention was confirmed. In Examples 1, 3 and 4, the carbon nanotube thin film was not coated with the polyvinyl alcohol solution, but the carbon nanotube thin film was not peeled off from the substrate.

Claims (8)

1. A step of adding a nanocarbon to a solvent not containing a binder agent to create a liquid containing nanocarbon,
   Applying the liquid containing the nanocarbon on a substrate to form a nanocarbon layer;
   A method for producing an electrode for a biosensor comprising a step of immobilizing an antibody or an enzyme on the nanocarbon layer.
2. In the manufacturing method of the electrode for biosensors of Claim 1,
   The said liquid is a manufacturing method of the electrode for biosensors which consists of the said nanocarbon and the said solvent.
3. In the manufacturing method of the electrode for biosensors of Claim 1 or 2,
   Before the step of adding the nanocarbon to the solvent,
   The manufacturing method of the electrode for biosensors which implements the process which wash | cleans the said nano carbon using an acid or an alkali solution.
4. In the manufacturing method of the electrode for biosensors in any one of Claims 1 thru | or 3,
   After the step of immobilizing the antibody or enzyme,
   The manufacturing method of the electrode for biosensors which implements the process of coat | covering the said nanocarbon layer with a polymer film.
5. In the manufacturing method of the electrode for biosensors in any one of Claims 1 thru | or 4,
   The said nano carbon is a manufacturing method of the electrode for biosensors which is a carbon nanotube.
6. In the manufacturing method of the electrode for biosensors in any one of Claims 1 thru | or 5,
   A method for producing an electrode for a biosensor, wherein the substrate and the nanocarbon layer are light transmissive.
7. A biosensor manufacturing method comprising the biosensor electrode manufacturing method according to claim 6,
   An alignment mark is formed at a position below the nanocarbon layer of the substrate,
   Including a step of mounting the biosensor electrode on a mounting substrate,
   In the step of mounting the biosensor electrode on a mounting substrate, the biosensor electrode is aligned using an alignment mark on the substrate of the biosensor electrode and an alignment mark formed on the mounting substrate, and the biosensor A biosensor manufacturing method for mounting an electrode for mounting on the mounting substrate.
8. In the manufacturing method of the biosensor according to claim 7,
   The nanocarbon layer as a working electrode,
   A biosensor manufacturing method comprising a step of forming a reference electrode and a counter electrode on the substrate.

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