JP7253169B2 - Substrate for SPR measurement and manufacturing method thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a substrate for surface plasmon resonance (SPR) measurement using a two-dimensional charged metal colloidal crystal, and a manufacturing method thereof. Here, the two-dimensional charged metal colloidal crystal refers to a single-layer regularly arranged structure (colloidal crystal) in which metal particles are arranged on a plane with a distance between the particles due to the electrical repulsive force acting between the particles. .

In recent years, various sensors using surface adsorption, such as medical sensors using antigen-antibody reaction, have been developed. These sensors require materials as transducers that convert the amount of surface adsorption into other signals with high sensitivity. As a material for such a sensor, we used surface plasmon resonance (SPR), which is a collective oscillation of free electrons in metals such as gold, silver, and platinum, which occurs when light is irradiated. Spectroscopic analysis has attracted attention. The principle of this technique is as follows.

In general, light does not couple with electron waves (plasmons), but on metal surfaces there are electron wave modes that couple with light due to surface-specific boundary conditions. This is called a surface plasmon. Methods of exciting surface plasmons include a method of forming a diffraction grating on a metal surface to couple light and plasmons, and a method of using evanescent waves. A sensor using surface plasmon resonance includes, for example, a sensor comprising a total reflection prism and a metal film that is formed on the surface of the prism and is in contact with a target substance. With such a configuration, the dielectric constant of the surface changes due to the small amount of adsorption of the antigen in the antigen-antibody reaction, and this changes the wavelength of the surface plasmon resonance. It can be performed.

By the way, propagating surface plasmons exist on a resonant metal surface that generates surface plasmons, but in metal particles, surface plasmons cannot propagate to the outside of the particles and are localized inside the particles. It is called. It is known that when localized surface plasmons are excited, a significantly enhanced electric field may be induced, and a highly sensitive localized plasmon resonance sensor utilizing this has been proposed. (Patent Document 1).

Furthermore, as shown in the schematic perspective view on the left side of Fig. 1, a metal-insulator-metal (MIM) type substrate for SPR measurement in which insulating spacers are interposed on the metal film and metal particles are placed on the spacers is also known (Non-Patent Document 1). According to this MIM-type SPR measurement substrate, a resonance phenomenon called "Fano resonance" occurs between the propagating plasmons of the metal film and the localized plasmons of the metal particles (see the graph on the right side of Fig. 1). Since the shape of the peak becomes a shape (sawtooth shape) in which the intensity changes sharply with a change in wavelength, a sensor with even higher sensitivity can be constructed.

JP-A-2000-356587

https://www.nature.com/articles/srep14419SCIENTIFIC REPORTS 5:14419 DOI:10,1038/srep14419

However, in the sensor using the SPR measurement substrate as described in Non-Patent Document 1, in order to achieve high sensitivity, the size of the metal particles and the gap between the metal particles must be controlled within a certain range. (see Figure 1). For this reason, its manufacture requires complicated pattern formation techniques such as the use of lithography to periodically arrange metal particles in a specific range vertically and horizontally. An increase was expected.

The present invention has been made in view of the above-mentioned conventional circumstances, and does not require a complicated pattern forming technique for controlling the arrangement of metal particles, is easy to manufacture, and has a high detection sensitivity. The problem to be solved is to provide a substrate for SPR measurement and a method for manufacturing the same, which can construct a surface plasmon sensor of this type.

The present inventors have developed a charged colloidal crystal made of metal as a method for eliminating the need for a pattern forming technique for controlling the arrangement of metal particles, which has been difficult to manufacture, in the conventional substrate for SPR measurement. thought of using it. A charged colloidal crystal is a state in which particles with a uniform particle size having a size of several nanometers to several micrometers are dispersed in a liquid such as water, and the particles are spaced apart to form an ordered structure that is regularly arranged periodically. are doing. Therefore, by selecting the manufacturing conditions (particle concentration, particle size, particle or medium refractive index, etc.), the arrangement of metal particles can be easily and automatically formed into colloidal crystal patterns without using complicated pattern formation techniques. can be formed. A charged colloidal crystal is characterized by gaps between particles, unlike an artificial opal-type colloidal crystal formed by contacting colloidal particles. Therefore, it is suitable for creating a structure in which the size of the metal particles and the gap between the metal particles are controlled within a certain range as described above (Non-Patent Document 1).

That is, the manufacturing method of the substrate for SPR measurement of the first invention is
an insulating member preparing step of preparing an insulating member having a positive or negative charge on the surface;
a colloidal crystal dispersion preparation step of preparing a charged colloidal crystal dispersion in which colloidal crystals made of metal colloid particles having a charge opposite to the surface charge of the insulating member are dispersed in a dispersion medium;
a surface forming step of bringing the charged colloidal crystal dispersion into contact with the insulating member to form a single-layer structure of metal colloidal crystals on the insulating member;
characterized by comprising

In the method for manufacturing a substrate for SPR measurement according to the first invention, first, as an insulating member preparation step, an insulating member having a positive or negative charge on the surface is prepared. On the other hand, as a colloidal crystal dispersion preparation step, a charged colloidal crystal dispersion is prepared in which colloidal crystals composed of metal colloid particles having a charge opposite to the surface charge of the insulating member are dispersed in a dispersion medium. Then, in the surface forming step, the charged colloidal crystal dispersion liquid is brought into contact with the insulating member, so that one layer of the charged colloidal crystal is adsorbed onto the insulating member by electrostatic attraction. In this way, a substrate for SPR measurement in which two-dimensional charged metal colloidal crystals are arranged on an insulating member at a predetermined lattice interval can be manufactured.

Therefore, according to the method for manufacturing a substrate for SPR measurement of the first invention, manufacturing is facilitated without requiring a complicated pattern forming technique such as lithography for controlling the arrangement of metal particles. In addition, since two-dimensional charged metal colloidal crystals are arranged on the insulating member with a predetermined lattice spacing, it is possible to manufacture a substrate for SPR measurement on which a localized surface plasmon sensor with high detection sensitivity can be constructed.

The method for manufacturing the substrate for SPR measurement of the second invention comprises:
An insulating member preparation step of preparing an insulating member;
a liquid layer forming step of forming, on the insulating member, a liquid layer composed of a charged colloidal crystal dispersion in which colloidal crystals composed of metal colloidal particles are dispersed in a dispersion medium;
After the liquid layer forming step, from one end side of the liquid layer, a charge preparation liquid capable of making the surface charge of the insulating member opposite in sign to the charge of the metal colloid particles is diffused, and the metal is spread on the insulating member. and a single-layer structure growth step for growing a single-layer structure of colloidal crystals.

In the method of manufacturing the substrate for SPR measurement of the second invention, first, an insulating member is prepared as an insulating member preparing step. On the other hand, in the liquid layer forming step, a liquid layer made of a charged colloidal crystal dispersion in which colloidal crystals made of metal colloidal particles are dispersed in a dispersion medium is formed on the insulating member. Furthermore, as a single layer structure growth step, after the liquid layer forming step, the surface charge of the insulating member can be made opposite in sign to the charge of the metal colloid particles from one end side of the liquid layer. The liquid is diffused to grow a monolayer structure of metal colloidal crystals (that is, two-dimensional charged metal colloidal crystals) on the insulating member. Such charge preparation liquids include 1) surfactants such as anionic surfactant solutions, cationic surfactant solutions, nonionic surfactant solutions, and amphoteric surfactant solutions; 2) hydrochloric acid, sulfuric acid, phosphoric acid nitrate, 3) alkali carbonates such as sodium carbonate; alkali hydrogen carbonates such as sodium hydrogen carbonate; alkali hydroxides such as sodium hydroxide; aqueous ammonia; and bases such as amines and pyridine.
In this way, by gradually growing the two-dimensional charged metal colloidal crystals on the insulating member by utilizing the diffusion phenomenon, the metal colloidal particles are regularly arranged at a certain distance on the insulating member, resulting in a two-dimensional crystal with fewer defects. A substrate for SPR measurement on which a charged colloidal crystal is constructed can be automatically formed.

Therefore, according to the method for manufacturing a substrate for SPR measurement of the second invention, a complicated pattern forming technique for controlling the arrangement of metal particles is not required, the manufacturing is easy, and the detection sensitivity is high. A substrate for SPR measurement can be manufactured on which a ready-made surface plasmon sensor can be constructed.

In the method for manufacturing a substrate for SPR measurement according to the second invention, the insulating member is made of a material whose surface charge changes depending on the ion concentration (for example, pH), and the charge preparation liquid has the surface charge of the insulating member with the opposite sign to the charge of the metal colloid particles. can be an acid or base capable of
In this case, by diffusing the acid or base from one end of the liquid layer, only one layer of the colloidal metal particles in the charged metal colloidal crystals is gradually adsorbed from one end to the other end of the liquid layer. . As a result, a two-dimensional charged colloidal crystal with fewer defects, in which the metal colloidal particles are regularly arranged at a constant distance on the insulating member, is constructed.
Examples of insulating materials whose surface charge changes with pH include glass, ceramics made of silicate, alumina, and functional groups whose surface charge changes with pH, such as amino groups, hydroxyl groups, sulfonic acid groups, and pyridyl groups. Examples include modified polymers.

Further, in the method for manufacturing the substrate for SPR measurement of the second invention,
The liquid layer forming step can be performed by the following first to third liquid layer forming steps. i.e.
1) A first liquid layer forming step of preparing a charged colloidal dispersion in which colloidal metal particles are dispersed in a dispersion medium;
2) a second liquid layer forming step of forming a liquid layer composed of a charged colloidal dispersion on the insulating member;
3) After the second step, a step comprising a third liquid layer forming step of diffusing a colloidal crystallization preparation liquid capable of colloidally crystallizing the charged colloidal dispersion from one end of the liquid layer.

According to this method, colloidal crystallization of the charged colloidal dispersion gradually grows from one end side due to the diffusion phenomenon in the liquid layer forming step, so colloidal crystals with fewer defects are present in the liquid layer. Further, as a single layer structure growth step, after the liquid layer forming step, the surface charge of the insulating member from one end side of the liquid layer can be made opposite in sign to the charge of the metal colloid particles. Allow the preparation to spread. As a result, each layer of the metal colloid crystal is gradually attracted onto the insulating member from one end side by electrostatic attraction. As a result, a two-dimensional charged metal colloid crystal with fewer defects, in which the metal colloid particles are regularly arranged at a constant distance on the insulating member, is constructed.

In the method for manufacturing a substrate for SPR measurement of the present invention, the insulating member is formed by forming an insulating film on a metal film, thereby forming an MIM (metal-insulator-metal) structure on the metal film. can be done.

As the positively charged insulating member, a silica film modified with a silane coupling agent having a positively charged dissociative group, or a film formed by adsorption of a positively charged polymer can be used. For example, at least one insulating film selected from a silica film modified with a silane coupling agent having an aminopropyl group and a film formed by adsorption of polyethyleneimine and poly(2-vinylpyridine) can be used. The inventors have confirmed that the SPR measurement substrate can be reliably produced by forming these insulating films.
Further, as the negatively charged insulating member, a film formed by adsorption of a negatively charged polymer can be used. Adsorption of anionic polymer electrolytes such as polyacrylic acid and polystyrenesulfonic acid and introduction of negative charges such as silanol groups can be used to impart a negative charge to the insulating film.

Also, the colloidal metal particles having a charge opposite in sign to the charge of the insulating member preferably have a sufficiently large amount of charge on the surface to form a charged colloidal crystal. For example, when it is desired to give a negative charge to a metal colloid particle, a compound (for example, mercaptoethanesulfonic acid) having a mercapto group and a charged group (such as a sulfonic acid group) is used, and the mercapto group is bound to the metal surface. Charged groups are introduced onto the particle surface. This is because the mercapto group forms a strong chemical bond with a metal such as gold. On the other hand, when it is desired to impart a positive charge to the metal colloid particles, the surface can be modified with a compound having a thiol group and a positive charge (for example, aminoethanethiol hydrochloride).

Also, the metal film may be a metal film formed on an insulating substrate. Examples of the insulating substrate include a glass substrate and a ceramic substrate.

Moreover, it is preferable that the metal film has a film thickness of 1 nm or more and 100 nm or less. If the film thickness is 100 nm or less, the intensity of the electric field due to the excitation of the localized surface plasmon becomes strong, so the sensitivity in the MIM type SPR measurement becomes high. Also, if the film thickness is 1 nm or more, the possibility that pinholes are formed in the metal film is reduced.

Also, the coefficient of variation of the particle size of the metal particles is preferably 20% or less. If the coefficient of variation of particle size is 20% or less, the colloidal crystal structure is more likely to be formed. Here, the coefficient of variation (CV) of particle size refers to the value of (standard deviation of particle size x 100/average particle size), more preferably 15% or less, more preferably 10% or less, and most preferably about 5% or less.

Moreover, it is preferable that the metal particles have an average particle diameter of 50 nm or more and 500 nm or less. If the average particle diameter of the metal particles is 500 nm or less, the sedimentation velocity in the liquid medium will be low, making it easier to form three-dimensional crystals. Also, if the average particle size of the metal particles is 50 nm or more, the thermal motion is not so vigorous, so that charged colloidal crystals are more likely to be generated.

Further, it is preferable to subject the insulating film to an alkali treatment before performing the surface forming step. By treating the insulating film with an alkali, 0 ⁻ groups are formed in the insulating film, which are gradually neutralized by carbonate ions in the air with the lapse of time, and the surface charge gradually increases to the positive side. Therefore, the growth of the two-dimensional charged metal colloidal crystal in the surface forming step is slowed down, and the two-dimensional charged metal colloidal crystal with few defects is formed.

By using the method for manufacturing a substrate for SPR measurement of the present invention, it is possible to easily manufacture a localized type substrate with high detection sensitivity without requiring a complicated pattern forming technique for controlling the arrangement of metal particles. A surface plasmon sensor can be constructed.
That is, the substrate for SPR measurement of the present invention is
A metal layer and an insulating layer are formed in this order on an insulating substrate, and a single-layer metal colloidal crystal made of metal particles is formed on the insulating layer,
The average particle diameter of the metal particles is 50 nm or more and 500 nm or less, and the distance between the metal particles is 50 nm or more and 1000 nm or less.

1 is a schematic diagram (left side) of a metal-insulator-metal (MIM) substrate for SPR measurement described in Non-Patent Document 1, and a graph (right side) showing the light absorption effect due to Fano resonance. FIG. 4 is a schematic diagram showing a manufacturing process of the substrate for SPR measurement of Embodiment 1; FIG. 2 is a schematic diagram showing that a charged colloidal crystal dispersion 2 having a negative charge is brought into contact with an insulating member 1 having a positive charge, and only one layer of the colloidal crystal lattice is attracted to the insulating member 1 by electrostatic attraction. FIG. 10 is a schematic diagram showing a manufacturing process of the substrate for MIM type SPR measurement of Embodiment 2; FIG. 10 is a schematic diagram showing a manufacturing process of the substrate for SPR measurement of Embodiment 3; 10 is a schematic cross-sectional view showing a method of diffusing the colloidal crystallization liquid 14 in the liquid layer 14 via the membrane filter 11 in the liquid layer forming step S32 in the manufacturing process of the substrate for SPR measurement of Embodiment 3. FIG. FIG. 3 is a schematic diagram showing the state when a gold-deposited glass plate is treated with MEPTMS and APTES. FIG. 2 is a schematic diagram showing charged colloidal gold particles whose surfaces are modified with MPS. 2 is an optical microscope photograph of the substrate for MIM-type SPR measurement of Example 1. FIG. 1 is a schematic perspective view of a substrate for MIM-type SPR measurement of Example 1. FIG. 1 is a schematic cross-sectional view of a substrate for MIM-type SPR measurement of Example 1. FIG. 10 is an optical micrograph of the substrate for MIM-type SPR measurement of Example 4. FIG. 1 is a schematic diagram of a colloidal crystal; FIG. FIG. 10 is a schematic diagram of a radial distribution function g(r) (left side) and adjacent colloidal gold particles (right side) for the substrate for MIM-type SPR measurement of Example 3; 4 shows the results of refractive index measurement of the substrate for MIM-type SPR measurement and the gold-deposited glass substrate of Example 2. FIG. 4 shows the measurement results of the refractive index on the substrate for MIM type SPR measurement of Example 3 and the substrate of Comparative Example 1. FIG. 1 is an optical microscope photograph of a silica crystal adsorption substrate (with NaOH treatment) of Reference Example 1. FIG. 4 is an optical microscope photograph of a silica crystal adsorption substrate (without NaOH treatment) of Reference Example 2. FIG. FIG. 3 is a schematic diagram of charge number control of a silica-coated gold-deposited glass plate treated with APTES. 2 is a schematic cross-sectional view of a diffusion device 20; FIG. 10 is an electron micrograph of the substrate for SPR measurement of Example 5. FIG.

(Embodiment 1)
Embodiment 1 is a method for manufacturing a substrate for SPR measurement according to the first invention. FIG. 2 shows a schematic diagram showing the manufacturing process thereof. Description will be made below with reference to FIG.
<Insulating member preparation step S11>
First, the insulating member 1 is prepared. As the insulating member 1, for example, an insulating glass substrate or a ceramic substrate can be used. These insulating members 1 usually have a negative surface potential due to silanol groups. 2-Vinylpyridine) or other polymer having a cationic group may be adsorbed on the surface to make the surface potential positive.

<Colloidal crystal dispersion preparation step S12>
On the other hand, in the colloidal crystal dispersion preparation step S12, a charged colloidal crystal dispersion 2 is prepared in which charged colloidal crystals composed of metal colloid particles having a negative (or positive) charge are dispersed in a dispersion medium. For example, in a colloidal dispersion of gold or the like, a compound (e.g., 3- Sodium mercapto-1-propanesulfonate (MPS), etc.) is added. When impurity ions are removed by adding an ion exchange resin, etc., the electrostatic repulsive force between the particles becomes sufficiently strong, a charged colloidal crystal structure is formed in the dispersion liquid, and the metal colloidal particles are arranged at predetermined intervals. A charged colloidal crystal dispersion 2 is prepared.

<Surface forming step S13>
Then, in the surface forming step S13, the negatively (or positively) charged colloidal crystal dispersion is brought into contact with the positively (or negatively) charged insulating member 1, and only one layer of the colloidal crystal lattice is insulated by electrostatic attraction. After being adsorbed to the member 1 (see FIG. 3), it is washed with a solvent such as water to wash away excess charged colloidal crystal dispersion. In this way, the substrate for SPR measurement of Embodiment 1, in which the two-dimensional charged metal colloidal crystals composed of the metal particles 3 are formed on the insulating member 1, is prepared.
According to the manufacturing method of Embodiment 1, the arrangement of metal particles can be controlled by forming two-dimensional charged colloidal crystals without using a complicated pattern forming technique, so manufacturing is easy, and A localized surface plasmon sensor with high detection sensitivity can be constructed.

(Embodiment 2)
Embodiment 2 is a method for manufacturing an MIM type substrate for SPR measurement. FIG. 4 shows a schematic diagram showing the manufacturing process thereof. Description will be made below with reference to FIG.
<Insulating film forming step S21>
A metal film substrate 7 is prepared by forming a metal film 6 on a substrate 5 made of insulating glass, ceramic, or the like by vapor deposition, sputtering, chemical plating, or the like. As the type of metal film, a noble metal such as gold that can exhibit strong plasmon resonance is suitable. Moreover, the thickness of the metal film is preferably set to a thickness (specifically, 1 to 100 nm) that allows efficient generation of surface plasmons. Then, a metal substrate 9 with an insulating film is produced by forming an insulating film 8 having a positive (or negative) charge on the metal film substrate 7 . As the insulating film 8 having a positive charge, for example, a silica layer is formed on a metal such as gold using a silane coupling agent having a mercapto group, and then the silica layer is coated with a second silane coupling agent. It can be formed by bonding an amino group or by adsorbing a polymer having a cationic group such as polyethyleneimine or poly(2-vinylpyridine) on the surface.

<Colloidal crystal dispersion preparation step S22>
On the other hand, as the colloidal crystal dispersion preparation step S22, in the same manner as in the colloidal crystal dispersion preparation step S12 of Embodiment 1, a charged colloid in which colloidal crystals composed of metal colloid particles having a negative (or positive) charge are dispersed in a dispersion medium A crystal dispersion liquid 2 is prepared.

<Surface forming step S23>
Then, in the surface forming step S23, the charged colloidal crystal dispersion liquid 2 was dropped onto the metal substrate 9 with an insulating film, and the negatively charged metal colloidal particles 10 were attracted to only one layer of the crystal lattice by electrostatic attraction. After that, the excess charged colloidal crystal dispersion 2 is washed away by washing with a solvent such as water. Thus, the substrate for MIM type SPR measurement of Embodiment 2 is prepared. In the MIM SPR measurement substrate of Embodiment 2, a resonance phenomenon called “Fano resonance” occurs between the propagating plasmons of the metal film and the localized plasmons of the metal particles, and this causes the shape of the absorption spectrum peak to change in wavelength. Since the shape (sawtooth shape) is such that the intensity changes sharply depending on the angle, it is possible to construct a sensor with even higher sensitivity.

(Embodiment 3)
Embodiment 3 is a method for manufacturing a substrate for SPR measurement according to the second invention. That is, in this method, a liquid layer composed of a charged colloidal crystal dispersion is formed on an insulating member, and a single layer structure of metal colloidal crystals (that is, a monolayer structure of metal colloidal crystals) is formed by diffusing the charge preparation liquid from one end of the liquid layer. A two-dimensional charged metal colloidal crystal) is grown to manufacture a substrate for SPR measurement. FIG. 5 shows a schematic diagram showing a manufacturing process diagram. Description will be made below with reference to FIG.
<Insulating member preparation step S31>
As the insulating members 11, two sheets of a surface-treated insulating member 11a made of a glass substrate or a ceramic substrate similar to the case of the first embodiment and an untreated insulating member 11b made of a glass substrate or a ceramic substrate without surface treatment were prepared, They face each other in parallel while maintaining a constant distance via spacers (not shown). Furthermore, the membrane filter 12 is inserted into one end side of the surface-treated insulating member 11a and the non-treated insulating member 11b (where the lower side is the surface-treated insulating member 11a) facing in parallel.

<Liquid Layer Forming Step S32>
Then, in the liquid layer forming step S32, the liquid layer 13 made of the charged colloidal crystal dispersion is formed. As a method for this purpose, the following two methods can be exemplified.
(Method 1)
A charged colloidal crystal dispersion liquid is prepared in which charged colloidal crystals composed of metal colloidal particles having a positive (or negative) charge are dispersed in a dispersion medium as used in the first embodiment. Then, the gap between the two insulating members 11 is filled with the charged colloidal crystal dispersion. Thus, the liquid layer 13 made of the charged colloidal crystal dispersion is formed in the gap between the two insulating members 11 .
(Method 2)
The liquid layer forming step S32 of Method 2 consists of the following three steps.
・First liquid layer forming step S321
A metal colloidal dispersion is prepared in which positively (or negatively) charged metal colloidal particles are dispersed in a solvent (in this dispersion, the metal colloidal particles are not colloidally crystallized).
・Second liquid layer forming step S322
Next, the gap between the two insulating members 11 is filled with the metal colloidal dispersion.
・Third liquid layer forming step S323
Then, as shown in FIG. 6, the membrane filter 12 side is connected to the reservoir tank 14 . Here, since the colloidal crystallized liquid 15 capable of colloidally crystallizing the metal colloidal dispersion is stored in the reservoir tank 14 , the colloidal crystallized liquid diffuses in the liquid layer 13 through the membrane filter 12 . do. Therefore, the metal colloidal dispersion in the liquid layer 13 colloidally crystallizes from the membrane filter 12 side toward the other end side.

<Single layer structure growth step S33>
Next, as shown in FIG. 5, the membrane filter 12 side of the surface-treated insulating member 11a and the non-treated insulating member 11b facing in parallel (where the surface-treated insulating member 11a is on the lower side) is connected to the reservoir tank 16. As shown in FIG. Here, a reservoir tank 16 stores a charge preparation liquid 17 capable of making the surface charge of the insulating member opposite in sign to the charge of the metal colloid particles, and the charge preparation liquid 17 is a membrane filter. 12 and diffuses in the liquid layer 15 . Therefore, the metal colloid particles in the liquid layer 15 are gradually adsorbed onto the surface-treated insulating member 11a by electrostatic attraction from the membrane filter 12 side. As a result, two-dimensional charged metal colloidal crystals 18 arranged at predetermined lattice intervals are formed. The two-dimensional charged metal colloidal crystal 18 formed in this way grows gradually by diffusion, so that it becomes a two-dimensional colloidal crystal with fewer defects, resulting in a localized surface plasmon sensor with even higher detection sensitivity.

EXAMPLES Hereinafter, examples in which the present invention is further embodied will be described in comparison with comparative examples.
(Example 1)
In Example 1, a substrate for MIM type SPR measurement was produced as follows.
・Insulating member preparation process Prepare a gold-deposited glass plate (manufactured by Kenis Co., Ltd.) in which a gold vapor-deposited layer is formed on a glass substrate. (hereinafter referred to as “Milli-Q water”) and stored in Milli-Q water. The gold-deposited glass plate thus washed was immersed in a 40 μM methanol solution of 3-Mercaptopropyltrimethoxysilane (MEPTMS) for 3 hours to modify the glass with MEPTMS (see FIG. 7(a)). This substrate was immersed in a 0.01 M NaOH aqueous solution for 2 hours to form a silica film. Next, the gold-deposited glass substrate treated with MEPTMS was immersed in a 1% solution of 3-Aminopropyltriethoxysilane (APTES) (solvent: 90% EtOH aqueous solution) for 1 hour (see FIG. 7B). After that, the gold-deposited glass substrate was taken out, and after lightly rinsing the surface with EtOH, it was dried in an oven at 80°C. In this way, an insulating film having a positive charge was produced on the deposited gold film.

• Colloidal Crystal Dispersion Preparation Step A charged colloidal dispersion in which gold colloid particles having a negative charge are dispersed in a dispersion medium was prepared by the following procedure. First, 3.92 mL of Milli-Q water and 0.08 mL of a 0.05 M solution of sodium 3-mercapto-1-propanesulfonate (MPS, Lot. #STBF3994V, manufactured by Sigma-Aldrich) were added to a centrifuge tube (Corning Life Sciences). It was dispersed by ultrasonic treatment for 5 minutes. Then, 4 mL of "Houtform AMS-Au" manufactured by Fuji Chemical Co., Ltd. (monodisperse gold colloid particle size: 244 nm, coefficient of variation of particle size: 12%, 1.3 wt%, Lot.1701) was added to this solution. Ultrasonic treatment was performed for 1 minute and centrifugation (1000 rpm, 30 minutes) was performed to precipitate colloidal gold particles having a sulfonic acid group as a negative charge (see FIG. 8). Furthermore, after removing the supernatant liquid, ultrapure water was newly added, and the operation of ultrasonically dispersing and centrifuging (1000 rpm, 30 minutes) was repeated three times, followed by washing with water. After modifying the colloidal gold particle surface with MPS in this way, it was purified with ion-exchange resin AG501-X8(D) type mixed (Bio-Rad Labs, CA, USA) for more than one day. A colloidal crystal dispersion was obtained in which was dispersed in the solvent.

・Surface formation process A small amount of a 60% aqueous solution of ethylene glycol (EG) purified with an ion exchange resin is dropped onto the surface of the substrate on which an insulating film with a positive charge is formed on the gold deposition film in the insulating material preparation process, and the cover glass is coated. After the liquid was developed and allowed to dry naturally, the charged colloidal gold crystal dispersion obtained in the colloidal crystal dispersion preparation process was dropped and allowed to stand for 10 minutes. -Q Stored in water. Thus, the substrate for MIM type SPR measurement of Example 1 was obtained.

(Example 2)
In Example 2, instead of 3-Mercaptopropyltrimethoxysilane (MEPTMS) and 3-Aminopropyltriethoxysilane (APTES) used in the insulating member preparation step of Example 1, polyethyleneimine was used.
That is, a gold-evaporated glass plate (manufactured by Kenis Co., Ltd.) having a gold vapor-deposited layer formed on a glass substrate was prepared, and the surface was washed with concentrated sulfuric acid. (manufactured by Film Wako Pure Chemical Industries, Ltd.), shaken for about 1 minute, shaken in Milli-Q water for 1 day, and stored in Milli-Q water.
Other steps are the same as in Example 1, and description thereof is omitted.

(Example 3)
In Example 3, poly(2-vinylpyridine) was used in place of polyethyleneimine in the insulating member preparation step of Example 2.
That is, a gold-evaporated glass plate (manufactured by Kenis Co., Ltd.) in which a gold vapor-deposited layer is formed on a glass substrate is prepared, the surface is washed with concentrated sulfuric acid, and then immersed in a 2 wt% aqueous solution of poly(2-vinylpyridine). After shaking for about 1 minute, it was shaken in Milli-Q water for 1 day and stored in Milli-Q water.
Other steps are the same as those in Examples 1 and 2, and description thereof is omitted.

(Example 4)
In Example 4, a gold-deposited glass plate was coated with MPTES in the same manner as in Example 1, and the surface was modified with APTES. After washing with water, colloidal gold crystals were adsorbed (that is, the only difference from Example 1 was that the substrate was treated with 0.01 M NaOH before adsorbing colloidal gold crystals).

(Comparative example 1)
In Comparative Example 1, the formation of an insulating film with poly(2-vinylpyridine) in Example 3 was performed on a glass substrate on which gold was not vapor-deposited, and a charged colloidal gold dispersion in which the surface of colloidal gold particles was modified with MPS was prepared. was brought into direct contact with a glass substrate, and colloidal gold particles were placed on the glass to obtain a gold particle-mounted glass substrate.

<Evaluation>
The MIM type SPR measurement substrates of Examples 1, 2, 3 and 4 and the gold particle-mounted glass substrate of Comparative Example 1 thus obtained were evaluated as follows.

(Observation with an optical microscope)
FIG. 9 shows an optical microscope photograph of the substrate for MIM type SPR measurement of Example 1. As shown in FIG. From this photograph, it was found that colloidal gold particles were regularly arranged to form colloidal crystals. In addition, the colloidal gold particles were observed only when the height of the sample stage in microscopic observation was set to a certain height, so it was found that the colloidal crystals of the gold colloidal particles were two-dimensional colloidal crystals consisting of only one layer. rice field. That is, as shown in FIG. 10, the gold colloidal particles were regularly arranged two-dimensionally in the substrate for MIM-type SPR measurement of Example 1, and only one layer was deposited. As shown in FIG. 11, the colloidal gold particles in the colloidal gold crystal are separated from each other by the charged colloid having a negative charge. I'm in. On the other hand, the insulating film has a positive charge and the gold particles have a negative charge, so the colloidal gold particles are firmly attached to the insulating film by electrostatic attraction.

It was also found that the MIM-type SPR measurement substrate of Example 2 similarly formed two-dimensional colloidal crystals in which gold particles were regularly arranged two-dimensionally.
Furthermore, it was found that the MIM-type SPR measurement substrate of Example 3 also formed a large colloidal single crystal in which gold particles were regularly arranged two-dimensionally.

Further, in the substrate for MIM type SPR measurement of Example 4, as shown in FIG. It was found to have fewer defects than 1-3. From this, it was found that a two-dimensional colloidal crystal with fewer defects was formed by treating the substrate with NaOH before adsorbing the gold colloidal crystal.
The reason is considered as follows. That is, the 0 ^- groups generated on the glass substrate by treating the substrate with NaOH are gradually neutralized by carbonate ions in the air with the lapse of time, so that the surface charge gradually increases to the positive side. For this reason, compared with Example 1 in which the glass substrate was not treated with NaOH, it is presumed that the adsorption of gold colloidal crystals slowed down and the number of defects decreased. The present inventors have observed that the surface potential of silica particles can be controlled over time by adding sodium bicarbonate, which is alkaline (Non-Patent Documents, Murai, Yamada, Yamanaka et al., Langmuir 2007, vol. 23, p. 7510-7517). It is strongly suggested that the above guess is correct.

(Measurement of radial distribution function)
For the substrate for MIM type SPR measurement of Example 3, the radial distribution function g(r) was obtained by the following method. The radial distribution function g(r) is the probability that another particle exists at a distance r from the center of a particle.

The density of particles inside the ring shown in FIG. 13 is given by the following equation (1). By dividing this by the average particle density ρ (the number of particles per unit area) of the system, the radial distribution function g(r) of the following equation (2) is obtained.

As a result of analyzing the microscopic image, the radial distribution function g(r) shown in the left graph of FIG. 14 was obtained. Since this graph has a sharp peak shape and a narrow half width, it was found that a regular colloidal crystal structure was formed. Moreover, since the distance of the first peak is the distance between the closest particles, it was found that the distance between the colloidal gold particles was 420 nm (see the schematic diagram on the right side of FIG. 14). The Lindemann ratio (LR) (ie, the ratio of the first peak half-width to the nearest interparticle distance) was 0.5.

(Refractive index measurement by MIM type SPR measurement substrate)
For the MIM type SPR measurement substrates of Examples 2 and 3, the refractive index of an ethylene glycol-water mixed solvent was measured.
That is, using an Abbe refractometer, a sample solution is placed on the stage of the refractometer maintained at 20 ° C. using a constant temperature water circulation type temperature jacket, and the refractive index is calculated using Snell's law from the angle of refraction of incident light. asked for The refractive index of water is 1.33 and that of ethylene glycol is 1.45, and the refractive index was adjusted by changing the concentration of ethylene glycol.

FIG. 15 shows the measurement results of the refractive index dependence of the absorption wavelength shift for the substrate for MIM type SPR measurement of Example 2 and the gold-deposited glass substrate.
In the MIM-type SPR measurement substrate of Example 2, in addition to a small maximum based on local surface plasmons (LSPR), a large maximum based on Fano resonance was observed (upper left graph in FIG. 15). Then, due to the change in the refractive index due to the change in the ratio of ethylene glycol (EG), the maximum peak shift at 700 nm based on the Fano resonance is 380 nm/RIU (refractive index change = ratio of wavelength change per 1). lithography substrate value of 300 nm/RIU. From this, it was found that the substrate for MIM type SPR measurement of Example 2 can measure the refractive index with high sensitivity.
On the other hand, in the glass only deposited with gold, which was measured as a comparison, surface plasmon resonance (SPR) was observed, but no significant peak shift was observed (see the lower left graph in FIG. 15).

Further, in the measurement of the refractive index of the substrate for MIM type SPR measurement of Example 3, a large peak based on Fano resonance was observed as shown in the central graph of FIG. Then, the peak shift RIU was a large value of 929 (see the graph on the right side of FIG. 16).
On the other hand, no peak due to Fano resonance was observed for the glass substrate of Comparative Example 1, in which only gold particles were placed. Then, the peak shift RIU was as small as 86 (see the graph on the right side of FIG. 16).

<Reference example>
In Example 4, in order to investigate the effect of treating the substrate with 0.01M NaOH before adsorbing the gold colloidal crystals, the following reference example was performed using negatively charged silica colloidal particles instead of the gold colloidal particles. was tested.

(Reference example 1)
In Reference Example 1, negatively charged silica colloidal particle crystals (Nippon Shokubai KE-50W, diameter: 500 nm, 2 vol%) were used instead of the gold colloidal particles in Example 4, and the substrate was treated with 0.01M NaOH. Other conditions are the same as in Example 4, and description thereof is omitted.

(Reference example 2)
In Reference Example 2, the NaOH treatment in Reference Example 1 was not performed. Others are the same as those of Reference Example 1, and the description thereof is omitted.

<Evaluation>
The samples of Reference Examples 1 and 2 obtained as described above were observed with an optical microscope. As a result, a two-dimensional crystal structure in which silica colloidal particles were regularly arranged was observed in the sample of Reference Example 1 subjected to NaOH treatment (see FIG. 17). On the other hand, in the sample of Reference Example 2, which was not treated with NaOH, disturbance of the adsorbed silica colloidal particles was observed (see FIG. 18). The above results indicate that the treatment of the substrate with NaOH gradually neutralizes the 0 ^- groups generated on the glass substrate by carbonate ions in the air over time, and the surface charge gradually increases to the positive side. Compared to the sample of Reference Example 1 in which the glass substrate was not treated with NaOH, it is explained that the silica colloidal crystal adsorbed more slowly and the number of defects decreased (see FIG. 19).

(Example 5)
Example 5 is an example of the first invention, in which a liquid layer made of a dispersion of charged colloidal gold crystals is formed on a microscope cover glass, and two-dimensional charged colloidal gold crystals are unidirectionally spread from one end of the liquid layer. was grown to prepare a substrate for SPR measurement. A detailed description will be given below.

・Insulating member preparation process A cover glass for a microscope (24 mm × 24 mm, manufactured by Matsunami Glass Industry Co., Ltd.) was prepared, soaked in concentrated sulfuric acid overnight or more and washed, and then prepared using a Milli-Q water maker. It was washed with pure water (hereinafter referred to as “Milli-Q water”) and stored in Milli-Q water. 3-aminopropyltriethoxysilcane (APTES, Lot.
711457, Shin-Etsu Kogyo Co., Ltd.) was immersed in a 1% ethanol (90%) aqueous solution for 1 hour to modify APTES on the cover glass. After that, the cover glass was taken out, the surface was lightly rinsed with EtOH, and dried in an oven at 40°C. Thus, a cover glass having a positive charge on its surface was produced.

- Colloidal Crystal Dispersion Preparation Step A charged colloidal dispersion in which gold colloid particles having a negative charge are dispersed in water was prepared by the following procedure. First, 3.92 mL of Milli-Q water and 0.08 mL of a 0.05 M solution of sodium 3-mercapto-1-propanesulfonate (MPS, Lot. #STBF3994V, manufactured by Sigma-Aldrich) were added to a centrifuge tube (Corning Life Sciences). It was dispersed by ultrasonic treatment for 5 minutes. Then, 4 mL of colloidal gold particles manufactured by Tanaka Kikinzoku Co., Ltd. (particle size: 150 nm, AU colloidal solution-SC, 0.0065 wt%) was added to this solution, ultrasonicated for 10 minutes, and centrifuged (1000 rpm, 30 minutes) to precipitate colloidal gold particles having a sulfonic acid group as a negative charge. Furthermore, after removing the supernatant liquid, ultrapure water was newly added, and the operation of ultrasonically dispersing and centrifuging (1000 rpm, 30 minutes) was repeated three times, followed by washing with water. Thus, the gold colloid particle surface was modified with MPS. In this colloidal gold dispersion, since the ionic strength is high, the electrostatic repulsion between particles is shielded and weakened, and colloidal crystals are not formed. However, when this dispersion is purified with an ion exchange resin AG501-X8(D) type mixed (Bio-Rad Labs, CA, USA), colloidal crystals in which colloidal gold particles are regularly arranged are dispersed in water as a colloidal crystal dispersion. became.

- Liquid Layer Forming Step Next, a diffusion device 20 shown in FIG. 20 was prepared. The diffusion device 20 is composed of a reservoir tank 21 and a diffusion cell 22 detachably connected to an opening 21 a provided on the side surface of the reservoir tank 21 . The reservoir tank 21 consists of a reservoir tank body 21b and a lid 21c. In the diffusion cell 22, cover glasses 23a (modified with APTES) and 23b (untreated) facing each other parallel to each other extend at intervals of 1 mm via spacers 24 made of silicon resin. Furthermore, a membrane filter 25 made of nitrocellulose (average pore size=0.05 μm) through which colloidal gold particles cannot pass is inserted at one end of the opening 21a side of the gap between the cover glasses 23a and 23b.

In the diffusion device 20 configured as described above, the diffusion cell 22 was removed from the diffusion device 20, and the negatively charged colloidal gold particles prepared in the colloidal crystal dispersion preparation step were placed in the gaps between the cover glasses 23a and 23b. A charged colloidal dispersion 26 dispersed in water was filled (the colloidal gold particles were not colloidally crystallized in this dispersion). Then, the membrane filter 25 side is connected to the reservoir tank 21 . Further, the lid 21c of the reservoir tank 21 was opened, and Milli-Q water 27 was stored in the reservoir tank main body 21b as a colloidal crystallization liquid capable of colloidally crystallizing the colloidal gold dispersion. The ions in the colloidal gold dispersion 26 pass through the membrane filter 26 and diffuse into the Milli-Q water 27 in the reservoir tank 21 . Therefore, the ion concentration in the colloidal gold dispersion 26 is diluted from the membrane filter 25 side toward the other end, and the ionic strength is lowered. Therefore, the repulsive force between gold colloidal particles gradually increased, and colloidal crystals grew from the membrane filter 25 side toward the other end side.

・Single layer structure growth process By performing the liquid layer formation process described above for 16 hours, the gold colloid particles in the gold colloid dispersion 26 are colloidally crystallized. A 10 μm sodium bicarbonate solution was stored as a charge control liquid for adsorption, and allowed to stand still for 3 hours to diffuse into the gold colloidal crystals. Thereafter, the inside of the diffusion cell 22 was washed with ultrapure water to wash away unadsorbed gold colloid particles. FIG. 21 is an electron microscope photograph of the substrate for SPR measurement in which two-dimensional gold colloidal crystals are thus formed on the cover glass 23a (image size is 30 μm×30 μm). From this photograph, it was found that two-dimensional gold colloidal crystals can also be prepared on a positively charged substrate by unidirectional adsorption of gold particles using the charge number adjustment by the ion concentration gradient.

The present invention is by no means limited to the description of the embodiments and examples of the invention. Various modifications are also included in the present invention without departing from the scope of the claims and within the scope that can be easily conceived by those skilled in the art.

The substrate for SPR measurement manufactured by the manufacturing method of the present invention has a two-dimensional colloidal crystal in which colloidal metal particles are regularly arranged at a certain distance on the insulating film, so that it has a localized surface with high detection sensitivity. Plasmon sensors can be constructed. Moreover, it does not require a complicated pattern forming technique for controlling the arrangement of the metal particles, is easy to manufacture, and enables significant cost reduction through mass production.

S11... Insulating member preparation step, S12... Colloidal crystal dispersion preparation step, S13... Surface forming step 1... Insulating member 2... Charged colloidal crystal dispersion, 3... Metal particles, 4... Substrate for SPR measurement S21... Insulating film formation Step S22 Colloidal crystal dispersion preparation step S23 Surface forming step 5 Substrate 6 Metal film 7 Metal film substrate 8 Insulating film 9 Metal substrate with insulating film 10 Colloidal metal particles S31 ... insulating member preparation step, S32 ... liquid layer forming step,
DESCRIPTION OF SYMBOLS 11... Insulating member, 12... Membrane filter, 13... Liquid layer, 14, 16... Reservoir tank, 15... Colloid crystallization liquid, 17... Charge preparation liquid, 18... Two-dimensional charged metal colloid crystal

Claims (15)

an insulating member preparing step of preparing an insulating member having a positive or negative charge on the surface;
a colloidal crystal dispersion preparation step of preparing a charged colloidal crystal dispersion in which colloidal crystals made of metal colloid particles having a charge opposite to the surface charge of the insulating member are dispersed in a dispersion medium;
a surface forming step of bringing the charged colloidal crystal dispersion into contact with the insulating member to form a single-layer structure of metal colloidal crystals on the insulating member;
A method for manufacturing an SPR measurement substrate, comprising: An insulating member preparation step of preparing an insulating member;
a liquid layer forming step of forming, on the insulating member, a liquid layer composed of a charged colloidal crystal dispersion in which colloidal crystals composed of metal colloidal particles are dispersed in a dispersion medium;
After the liquid layer forming step, from one end side of the liquid layer, a charge preparation liquid capable of making the surface charge of the insulating member opposite in sign to the charge of the metal colloid particles is diffused, and the metal is spread on the insulating member. a single-layer structure growth step for growing a single-layer structure of colloidal crystals;
A method for manufacturing an SPR measurement substrate, comprising: The insulating member is made of a material whose surface charge changes depending on the ion concentration,
4. The method for manufacturing a substrate for SPR measurement according to claim 3, wherein the charge preparation liquid is an acid or a base capable of making the surface charge of the insulating member opposite in sign to the charge of the metal colloid particles. . The liquid layer forming step includes
a first liquid layer forming step of preparing a charged colloidal dispersion in which colloidal metal particles are dispersed in a dispersion medium;
a second liquid layer forming step of forming a liquid layer composed of a charged colloidal dispersion on the insulating member;
After the second step, a third liquid layer forming step of diffusing a colloidal crystallization preparation liquid capable of colloidally crystallizing the charged colloidal dispersion from one end of the liquid layer;
4. The method for manufacturing a substrate for SPR measurement according to claim 2 or 3, characterized by comprising: 5. The method for manufacturing a substrate for SPR measurement according to claim 1, wherein the insulating member has an insulating film formed on a metal film. 6. The method for manufacturing a substrate for SPR measurement according to claim 1, wherein said insulating member is made of a polymer. 7. The method of manufacturing a substrate for SPR measurement according to claim 6, wherein the polymer is at least one selected from polyethyleneimine and poly(2-vinylpyridine). 7. The method for manufacturing a substrate for SPR measurement according to claim 1, wherein the colloidal metal particles have dissociative groups on their surfaces. 6. The method of manufacturing a substrate for SPR measurement according to claim 5, wherein the metal film is a metal film formed on an insulating substrate. 10. The method of manufacturing a substrate for SPR measurement according to claim 5, wherein the metal film has a film thickness of 1 nm or more and 100 nm or less. 11. The method for producing a material for SPR measurement according to any one of claims 1 to 10, wherein the metal particles have a coefficient of variation of particle diameter of 20% or less. 12. The method for manufacturing a substrate for SPR measurement according to claim 1, wherein the metal particles have an average particle diameter of 50 nm or more and 500 nm or less. 13. The method of manufacturing a substrate for SPR measurement according to any one of claims 1 to 12, wherein the insulating member is treated with an alkali before performing the surface forming step. A metal layer and an insulating layer are formed in this order on an insulating substrate, and a single-layer metal colloidal crystal made of metal particles is formed on the insulating layer,
A substrate for SPR measurement, wherein the average particle diameter of the metal particles is 50 nm or more and 500 nm or less, and the distance between the metal particles is 50 nm or more and 1000 nm or less. 15. The substrate for SPR measurement according to claim 14, wherein the coefficient of variation of particle diameter of said metal particles is 20% or less.

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