SPR measurement substrate and method for manufacturing same

Abstract

[Problem] To provide: an MIM-type SPR measurement substrate that does not require a complicated pattern forming technique, that is easily manufactured, and that has high detection sensitivity; and a method for manufacturing the same. [Solution] A substrate on which a metal oxide layer having 4 or more and less than 8 isoelectric points is formed on a metal film is prepared (substrate preparation step S1). A three-dimensional charged colloidal crystal dispersion, in which charged colloidal crystals composed of metal particles are dispersed in a dispersion medium, and the sign of the surface charge of the charged colloidal crystals is the same as that of the surface charge of the metal oxide layer, is prepared (colloidal crystal dispersion preparation step S2). After the charged colloidal crystal dispersion is brought into contact with the metal oxide layer, the surface charge of the metal oxide layer is adjusted so that the sign thereof is different from that of the surface charge of the three-dimensional charged colloidal crystals, and two-dimensional charged metal colloidal crystals are transferred onto the metal oxide layer (transfer step S3).

Description

SPR measurement substrate and method for manufacturing same

[0001] The present invention relates to a metal-insulator-metal (MIM)-type substrate for surface plasmon resonance (SPR) measurements that utilizes two-dimensional charged metal colloidal crystals, and a method for manufacturing the same. Here, the term "two-dimensional charged metal colloidal crystals" refers to a monolayer regularly arranged structure (colloidal crystal) in which metal particles are arranged at a distance on a plane due to the electrical repulsive forces acting between the particles.

[0002] In recent years, various sensors that utilize surface adsorption have been developed, such as medical sensors that utilize antigen-antibody reactions. These sensors require a transducer material that can convert the amount of surface adsorption into another signal with high sensitivity. As a material for such sensors, spectroscopic analysis using SPR, which is the collective oscillation of free electrons in metals such as gold, silver, and platinum that occurs when light is irradiated on these metals, has attracted attention. The principle of this technology is as follows.

[0003] Generally, light does not couple with electron waves. However, on metal surfaces, a mode that couples with light occurs due to the boundary conditions specific to the surface. This is called surface plasmon. Methods for exciting surface plasmon include forming a diffraction grating on the metal surface to couple light with plasmons, and using evanescent waves. An example of a sensor that utilizes surface plasmon resonance is a sensor consisting of a total internal reflection prism and a metal film formed on the surface of the prism that contacts a target substance. With this configuration, the dielectric constant of the surface changes depending on the minute amount of antigen adsorption in an antigen-antibody reaction, which in turn changes the wavelength of the surface plasmon resonance. Therefore, minute amounts of antigen adsorption can be sensed from the change in resonance wavelength.

[0004] Meanwhile, propagating surface plasmons exist on metal surfaces that generate surface plasmons, but in metal particles, surface plasmons cannot propagate outside the particle and are localized inside, so they are called "localized surface plasmons." It is known that when localized surface plasmons are excited, a significantly enhanced electric field can be induced, and a highly sensitive localized plasmon resonance sensor utilizing this has been proposed (Patent Document 1).

[0005] Furthermore, as shown in the schematic perspective view on the left side of Figure 1, a MIM-type SPR measurement substrate is also known, in which an insulating spacer is interposed on a metal film and metal particles are placed on the spacer (Non-Patent Document 1). With this MIM-type SPR measurement substrate, a resonance phenomenon called "Fano resonance" occurs between the propagating plasmon of the metal film and the localized plasmon of the metal particles (see the graph on the right side of Figure 1), which causes the shape of the absorption spectrum peak to change sharply with wavelength (sawtooth shape), enabling the construction of an even more sensitive sensor.

[0006] However, in order to achieve high sensitivity in sensors using MIM-type SPR measurement substrates, it is necessary to control the size of the metal particles and the gaps between the metal particles within a certain range (see Figure 1). This poses a problem in that their manufacture requires complex pattern formation techniques, such as the use of lithography to periodically arrange the metal particles in a specific range.

[0007] To solve this problem, the present inventors have succeeded in producing an MIM-type substrate for SPR measurement by contacting a three-dimensional metal colloidal crystal dispersion with a substrate and transferring two-dimensional metal colloidal crystals onto the substrate (Patent Document 2). This method makes it possible to easily produce an MIM-type substrate for SPR measurement without using complex patterning techniques.

[0008] JP 2000-356587 A JP 2020-034543 A

[0009] https: / / www.nature.com / articles / srep14419SCIENTIFIC REPORTS 5:14419 DOI:10,1038 / srep14419

[0010] However, the method for manufacturing an MIM type SPR measurement substrate described in Patent Document 2 above has been required to achieve even higher sensitivity.

[0011] The present invention has been made in view of the above-mentioned conventional situation, and aims to provide a highly sensitive MIM-type SPR measurement substrate that is easy to manufacture without requiring complex pattern formation technology, and a method for manufacturing the same.

[0012] The method for manufacturing an MIM-type SPR measurement substrate of the present invention is characterized by comprising: a substrate preparation step of preparing a substrate having a metal oxide layer having an isoelectric point of 4 or more and 8 or less formed on a metal film; a colloidal crystal dispersion preparation step of preparing a three-dimensional charged colloidal crystal dispersion in which charged colloidal crystals made of metal particles are dispersed in a dispersion medium, and the surface charge of the charged colloidal crystals has the same sign as the surface charge of the metal oxide layer; and a transfer step of contacting the metal oxide layer with the charged colloidal crystal dispersion, and then adjusting the surface charge of the metal oxide layer so that it has a different sign from the surface charge of the three-dimensional charged colloidal crystal, and transferring a two-dimensional charged metal colloidal crystal onto the metal oxide layer.

[0013] In the method for manufacturing an MIM-type SPR measurement substrate of the present invention, a substrate is prepared in which a metal oxide layer having an isoelectric point of 4 to 8 is formed on a metal film (substrate preparation step). A three-dimensional charged colloidal crystal dispersion is also prepared in which charged colloidal crystals made of metal particles are dispersed in a dispersion medium, and the surface charge of the charged colloidal crystals has the same sign as the surface charge of the metal oxide layer (colloidal crystal dispersion preparation step). Then, in the transfer step, the charged colloidal crystal dispersion is brought into contact with the metal oxide layer, and the surface charge of the metal oxide layer on the substrate is adjusted to have a different sign from the surface charge of the three-dimensional charged colloidal crystals. Because the surface charge of the colloidal crystals has the same sign as the surface charge of the metal oxide layer when the charged colloidal crystal dispersion is brought into contact with the metal oxide layer, the colloidal crystals do not adsorb to the metal oxide layer, and three-dimensional charged colloidal crystals grow. Then, by adjusting the surface charge of the metal oxide layer so that it has a different sign from the surface charge of the three-dimensional charged colloidal crystal, an electrostatic attraction occurs between the grown three-dimensional charged colloidal crystal and the metal oxide layer, and the two-dimensional colloidal crystal is transferred onto the metal oxide layer.

[0014] In the two-dimensional colloidal crystal formation process, adjusting the surface charge of the metal oxide layer to have a different sign from the surface charge of the three-dimensional charged colloidal crystals can be easily achieved by adjusting the pH of the three-dimensional charged colloidal crystal dispersion within a pH range spanning the isoelectric point. The concentration of acid or alkali required to achieve a pH of the dispersion spanning the isoelectric point of 4 or greater than 8 is lower than when spanning an isoelectric point below 4 or above 8, allowing for a lower salt concentration in the three-dimensional charged colloidal crystal dispersion. As a result, the electric double layer becomes thicker, preventing aggregation of colloidal particles and melting of the colloidal crystal structure. This allows for smooth transfer of the two-dimensional colloidal crystal to the substrate and fewer defects in the crystal structure. Specific methods for changing the pH of a dispersion of three-dimensional charged colloidal crystals include 1) adding an acid or alkali solution, 2) using a cation exchange resin or anion exchange resin to adsorb hydrogen ions or hydroxide ions, and 3) absorbing a gaseous acid (e.g., hydrogen chloride gas) or alkali (e.g., ammonia gas or carbon dioxide gas), etc. When carbon dioxide gas is used, carbon dioxide gas contained in the atmosphere may be used.

[0015] According to the inventors' test results, by keeping the pH change of the dispersion of three-dimensional charged colloidal crystals in the two-dimensional colloidal crystal formation process to 4 or less and the salt concentration to 150 μM or less, the transfer of the two-dimensional colloidal crystals to the substrate is carried out more smoothly, and defects in the crystal structure are reliably reduced.

[0016] The isoelectric point of the metal oxide layer is preferably in the range of 4 to 8 inclusive, with 4.5 to 7.5 being more preferred, from the viewpoint of reducing defects in the two-dimensional colloidal crystal structure. Examples of metal oxide layers having an isoelectric point of 4 to 8 inclusive include titania layers with an isoelectric point of approximately 6 and alumina layers with an isoelectric point of 8. Furthermore, layers made of metal composite oxides containing a mixture of at least two metal elements, Ti, Si, and Al, can also be used. Titania-silica, alumina-silica, and titania-alumina are particularly suitable for use in order to obtain an isoelectric point of 4 to 8 inclusive. The metal composite oxide can be more precisely controlled to achieve a predetermined isoelectric point by varying the content ratio of each metal.

[0017] Furthermore, the metal oxide layer can be easily obtained by the hydrolysis polycondensation method (the so-called sol-gel method) of metal alkoxides. The sol-gel method allows for more precise control of the isoelectric point of the metal oxide layer by adjusting the ratio of the multiple metal alkoxides used.

[0018] The coefficient of variation of the particle size of the metal particles that make up the three-dimensional charged colloidal crystal is preferably 20% or less. If the coefficient of variation of the particle size is 20% or less, the colloidal crystal structure is more easily formed and defects in the crystal structure are reduced. Here, the coefficient of variation (CV) of the particle size refers to the value of (standard deviation of particle size x 100 / average particle size), and is preferably 15% or less, more preferably 10% or less, and most preferably about 5% or less.

[0019] Furthermore, the metal particles constituting the three-dimensional charged colloidal crystal preferably have an average particle size of 50 nm or more and 500 nm or less. If the average particle size of the metal particles is 500 nm or less, the sedimentation rate in the dispersion liquid becomes slower, making it easier to generate three-dimensional charged colloidal crystals. Furthermore, if the average particle size of the metal particles is 50 nm or more, the thermal motion is not so intense, making it easier to generate charged colloidal crystals.

[0020] Furthermore, the thickness of the metal film is preferably 1 nm or more and 100 nm or less. If the film thickness is 100 nm or less, the strength of the electric field due to the excitation of localized surface plasmons is increased, thereby increasing the sensitivity in MIM-type SPR measurements. Furthermore, if the film thickness is 1 nm or more, the possibility of pinholes occurring in the metal film is reduced. A film thickness of 5 nm or more and 50 nm or less is even more preferable.

[0021] The MIM-type SPR measurement substrate of the present invention has a metal oxide layer having an isoelectric point of 4 or more and less than 8 formed on the substrate, and a two-dimensional colloidal crystal layer made of metal particles formed on the metal oxide layer. This MIM-type SPR measurement substrate can be produced by the two-dimensional colloidal crystal production method of the present invention.

[0022] The metal particles constituting the two-dimensional colloidal crystal layer may be colloidal metal particles of the platinum group, such as colloidal gold particles or colloidal platinum particles, or may be colloidal silver particles.

[0023] By binding an antibody or antigen to the metal oxide layer and / or the metal particles in the MIM-type SPR measurement substrate of the present invention, it can be used as the immunosensor of the first invention. When this immunosensor is immersed in an antigen (or antibody) solution as the analyte, the antigen (or antibody) binds to the antibody (or antigen), causing a change in dielectric constant. This change in dielectric constant can be interpreted as a change in SPR wavelength, allowing the concentration of the antigen (or antibody) to be measured. Note that while the antibody (or antibody) may be bound to either the metal oxide layer or the metal particles, it is preferable to bind it to both the metal oxide layer and the metal particles in order to achieve a more sensitive immunosensor.

[0024] If the metal oxide layer of the MIM-type SPR measurement substrate is susceptible to hydrolysis, a tetraalkoxysilane hydrolysate layer may be provided on the surface of the metal oxide layer. Specifically, the immunosensor of the second invention comprises a metal oxide layer having an isoelectric point of 4 to 8 formed on a metal film, a two-dimensional colloidal crystal layer composed of metal particles formed on the metal oxide layer, a tetraalkoxysilane hydrolysate layer formed on the surface of the metal oxide layer, and antibodies or antigens bound to the tetraalkoxysilane hydrolysate layer and / or the metal particles. Since the pH of body fluids is approximately 7.4, an immunosensor usable at around pH 7.4 is preferred. If the metal oxide layer is susceptible to hydrolysis at around pH 7.4, protecting the metal oxide layer with a tetraalkoxysilane hydrolysate layer can result in an immunosensor usable at around pH 7.4. Tetraalkoxysilane is a general term for compounds in which four alkoxy groups are bonded to silicon, such as tetramethoxysilane and tetraethoxysilane. The alkoxy group of tetraalkoxysilane is easily hydrolyzed and forms a siloxane bond via the silanol group, so by covering the metal oxide layer, the hydrolysis of the metal oxide layer can be prevented.

[0025] This figure shows a schematic diagram (left) of an MIM (metal-insulator-metal) SPR measurement substrate described in Non-Patent Document 1, and a graph (right) showing the absorption effect due to Fano resonance. This figure shows a schematic diagram illustrating the manufacturing process for an MIM-type SPR measurement substrate. This figure shows a schematic cross-sectional view of a gold-deposited glass plate when the surface is treated with MEPTMS and then titanium alkoxide. This figure shows a graph indicating whether or not silica colloid particles are adsorbed to the surface-modified glass substrates of Test Examples 1 to 4, in which the mixture ratio of TTIP and TEOS was varied, at various HCl concentrations. This figure shows the results of isoelectric point measurements for the surface-modified glass substrates of Test Examples 1 to 4, in which the mixture ratio of TTIP and TEOS was varied. This figure shows a graph indicating whether or not silica colloid particles are adsorbed to the surface-modified glass substrates of Test Examples 5 to 8, in which the mixture ratio of ALIP and TEOS was varied, at various HCl and NaOH concentrations. This figure shows the results of isoelectric point measurements for the surface-modified glass substrates of Test Examples 5 to 8, in which the mixture ratio of ALIP and TEOS was varied. This figure shows a graph indicating the baking temperature and whether or not TTIP surface-modified glass substrates are adsorbed to the substrate at various HCl concentrations. 1 is a graph showing the presence or absence of adsorption of silica colloidal particles to the surface-modified glass substrates of Test Examples 9 to 12 at various pH levels, in which the mixing ratio of TTIP and ALIP was varied.

[0034] FIG. 1 is a graph showing the results of measuring the isoelectric points of the surface-modified glass substrates of Test Examples 9 to 12, in which the mixing ratio of TTIP and ALIP was varied.

[0035] FIG. 1 is an electron microscope photograph of the surface of the MIM-type SPR measurement substrate of Example 2.

[0036] FIG. 1 is an electron microscope photograph of the surface of the MIM-type SPR measurement substrate of Comparative Example 1.

[0037] FIG. 1 is a schematic cross-sectional view of a gold-deposited glass plate surface-treated with MEPTMS and APTES, in that order.

[0038] FIG. 1 is a graph showing the results of SPR measurement of the MIM-type SPR measurement substrate and LSPR measurement substrate of Example 1.

[0039] FIG. 2 is a graph showing the relationship between the SPR peak shift (Δλ) and the number of layers for the MIM-type SPR measurement substrate and LSPR measurement substrate of Example 1.

[0039] FIG. 2 is a graph showing the results of SPR measurement of the MIM-type SPR measurement substrates of Example 1 and Comparative Example 1. 1 is a graph showing the relationship between the SPR peak shift (Δλ) and the number of layers in the MIM-type SPR measurement substrates of Example 1 and Comparative Example 1. FIG. 2 is a perspective view showing an example of a calculation model for an MIM structure. FIG. 3 is a graph showing simulation results for interparticle distance dependency. FIG. 4 is a graph comparing FDTD simulation and experimental values.It is a graph showing the result of performing FDTD simulation on the influence of the thickness of the gold evaporation layer. It is the model used in the FDTD simulation. It is a graph showing the FDTD simulation result when protein is adsorbed, using the model of FIG. 22. It is the model used in the FDTD simulation. It is a graph showing the FDTD simulation result when protein is adsorbed, using the model of FIG. 24. (Particle center-to-center distance 330 nm) It is a graph showing the FDTD simulation result when protein is adsorbed, using the model of FIG. 25. (Particle center-to-center distance 560 nm) It is a graph showing the result of simulating the influence of the distance between gold particles on the plasmon peak by the FDTD method.

[0026] (Embodiment) Figure 2 shows a manufacturing process diagram for an MIM-type SPR measurement substrate according to an embodiment. The following description will be given with reference to Figure 2. <Substrate Preparation Step S1> A metal film-coated substrate 3 is prepared by forming a metal film 2 on a substrate 1 made of insulating glass, ceramic, or the like by vapor deposition, sputtering, chemical plating, or other techniques. The metal film 2 is preferably made of a noble metal such as gold, which exhibits strong plasmon resonance. The thickness of the metal film 2 is preferably a thickness that allows efficient generation of surface plasmons (specifically, 1 to 100 nm). An insulating film-coated metal substrate 5 is then prepared by forming a metal oxide layer 4 having an isoelectric point of 4 or more but less than 8 on the metal film 2. The following methods can be used to form the insulating film-coated metal substrate 5 (see Figure 3). (1) An insulating substrate such as a glass substrate is prepared, and a platinum group metal film (e.g., a gold film, a platinum film, etc.) is formed on the metal film 2 by vacuum deposition, sputtering, or chemical plating. (2) The metal coating is surface-modified using an organic compound having a thiol group and an anionic functional group (e.g., 3-mercapto-1-propanesulfonic acid sodium salt). (3) A metal oxide film having an isoelectric point of 4 or more but less than 8 is then formed by hydrolysis and dehydration (sol-gel method) of titanium alkoxide, aluminum alkoxide, or silicon alkoxide. Specifically, the substrate 1 is immersed in a solution containing a metal alkoxide, hydrolyzed with acid or water, then removed from the solution and heated to undergo polycondensation to form a titania layer. Alternatively, the substrate can be immersed in a solution containing a mixture of multiple metal alkoxides, removed from the solution, and heated to undergo polycondensation to form a metal composite oxide with various ratios depending on the solution composition. The film thickness can be controlled by controlling the removal speed when removing the substrate 1 from the immersion solution. The isoelectric point can be adjusted by appropriately adjusting the mixing ratio of these alkoxides. Alternatively to the sol-gel method, dry methods such as vacuum deposition, sputtering, and CVD can also be used. In these dry methods, titania, silica, or alumina is used as the metal source, and a method of reacting Ti and oxygen in the system to form titania can also be used.

[0027] <Colloidal Crystal Dispersion Preparation Step S2> Meanwhile, in the colloidal crystal dispersion preparation step S2, a charged colloidal crystal dispersion 6 is prepared in which charged colloidal crystals composed of metal colloidal particles having a surface charge opposite in sign to that of the metal oxide layer 4 are dispersed in a dispersion medium. The metal colloidal particles preferably have a sufficiently large surface charge to form charged colloidal crystals. For example, if a negative charge is desired on the metal colloidal particles, a compound (e.g., mercaptoethanesulfonic acid) having a mercapto group and a charged group (e.g., sulfonic acid group) is used to bond the mercapto group to the metal surface, thereby introducing the charged group onto the particle surface. This is because the mercapto group forms a strong chemical bond with metals such as gold. On the other hand, if a positive charge is desired on the metal colloidal particles, this can be achieved by surface modification with a compound (e.g., aminoethanethiol hydrochloride) having a thiol group and a positive charge. Methods for adjusting the surface charge of metal colloidal particles to a predetermined level include adding an acid, alkali, or salt, or using a cation exchange resin or anion exchange resin to adsorb cations or anions. Removing impurity ions by adding an ion exchange resin or the like strengthens the electrostatic repulsive force between particles sufficiently, forming a charged colloidal crystal structure in the dispersion, and facilitating the formation of a three-dimensional charged colloidal crystal in which metal colloidal particles are arranged at a predetermined interval.

[0028] <Transfer Step S3> In the transfer step S3, the pH of the charged colloidal crystal dispersion 6 is adjusted as follows: 1) the pH of the charged colloidal crystal dispersion 6 is adjusted so that the surface charge of the metal oxide layer 2 and the surface charge of the metal colloid particles have the same sign, allowing three-dimensional charged colloidal crystals to grow sufficiently; and 2) the pH is readjusted so that the surface charge of the metal oxide layer 4 has the opposite sign to the surface charge of the metal colloid particles, thereby electrostatically adsorbing the three-dimensional charged colloidal crystals to the surface of the metal oxide layer 4. This two-stage pH adjustment prevents the metal colloidal particles from randomly adsorbing to the metal oxide layer 4 before the three-dimensional charged colloidal crystals are formed, thereby reducing defects in the crystal structure of the two-dimensional charged metal colloidal crystals 7. Alternatively, the pH can be readjusted by first removing carbon dioxide from the charged colloidal crystal dispersion 6 to increase the pH, and then later lowering the pH by absorbing carbon dioxide from the air. Finally, the excess three-dimensional charged colloidal crystal dispersion 6 is washed away with a solvent such as water, and the two-dimensional charged metal colloidal crystals 7 are transferred onto the metal oxide layer 4. In this way, the MIM-type SPR measurement substrate 8 of the embodiment is prepared.

[0029] In the manufacturing method of the MIM-type SPR measurement substrate of the embodiment, the isoelectric point of the metal oxide layer 4 is set to 4 or more and 8 or less. Therefore, by changing the pH of the three-dimensional charged colloidal crystal dispersion 6 within the range of 4 or more and 8 or less, the surface charge of the three-dimensional charged colloidal crystal can be made opposite to the surface charge of the metal oxide layer 4, resulting in the three-dimensional charged colloidal crystal being electrostatically adsorbed to the metal oxide layer 4. This prevents the melting of the three-dimensional charged colloidal crystal and particle aggregation due to increased ionic strength, allowing two-dimensional charged metal colloidal crystals 7 with fewer defects in the crystal structure to be transferred onto the metal oxide layer 66. Generally, a particle-to-surface distance of 300 to 600 nm is required for substrates utilizing surface plasmon resonance (SPR) (see Scientific Reports, Volume 5, Article number: 14419 (2015)). The inventors have discovered that preparing three-dimensional charged colloidal crystals with such wide interparticle distances is effective when the three-dimensional charged colloidal crystals are prepared under conditions of low salt concentration, such as 150 μM or less. By using the manufacturing method of the above embodiment, three-dimensional charged colloidal crystals can be prepared under conditions of low salt concentration, such as 150 μM or less, and the three-dimensional charged colloidal crystals can be fixed to the metal oxide layer 4 under low salt concentration conditions. As a result, the electric double layer becomes thicker, preventing aggregation of colloidal particles and melting of the colloidal crystal structure. This allows for smooth transfer of the two-dimensional colloidal crystals to the substrate and reduces defects in the crystal structure.

[0030] By binding antibodies (or antigens) to the metal oxide layer and / or metal colloid particles in the MIM-type SPR measurement substrate of the embodiment, it can be used as an immunosensor. The binding method can be chemical or physical. A specific example of chemical modification is to introduce carboxyl groups into the metal oxide layer and / or metal colloid particles using an aqueous silane coupling agent that has a carboxylic acid group in the organic functional group, and then bind the antibody (or antigen) to the carboxyl group using an amine coupling method, thereby easily chemically modifying the antibody. When this immunosensor is immersed in an antigen solution as the analyte, the antigen binds to the antibody, causing a change in the dielectric constant. This change in dielectric constant can be interpreted as a change in the SPR wavelength, allowing the concentration of the antigen (or antibody) to be measured.

[0031] <Relationship between insulating film composition and isoelectric point> In the MIM-type SPR measurement substrate of the present invention, it is necessary to control the isoelectric point of the metal oxide layer in the range of 4 or more and less than 8. For this reason, glass substrates with metal oxide layers formed thereon were prepared in the following Test Examples 1 to 12, and the relationship between the insulating film composition and isoelectric point was investigated. The reagents used to form the metal oxide layer are as follows: titanium tetraisopropoxide (abbreviation: TTIP): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. tetraethoxysilane (abbreviation: TEOS): manufactured by Shin-Etsu Chemical Co., Ltd. aluminum isopropoxide (abbreviation: ALIP): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0032] Test Examples 1-4 Preparation of Titania and Titania-Silica Composite Oxide Layer-Coated Glass Substrates 50 μL of TEOS, 92.5 mL of ethanol, 4.5 mL of 0.1 M HCl, and 5.6 μL of Milli-Q water were mixed in a glass bottle and incubated at 40°C for 10 minutes to prepare a TEOS hydrolyzed solution. EtOH, TTIP, the TEOS hydrolyzed solution, 0.1 M HCl solution, and purified water prepared using a Milli-Q water maker (hereafter referred to as "Milli-Q water") were then added to the glass bottle in the proportions shown in Table 1 and stirred at 300 rpm for 20 hours. The reaction solution was transferred to a polypropylene staining tray (Fisher Scientific), and a glass substrate (NEO micro cover glass, 24 mm × 60 mm, Matsunami Glass Industry) was immersed in the solution for 1 hour. After immersion, the glass substrate was taken out and kept in an oven at 50°C for 1 hour, and then the glass surface was washed with Milli-Q water and dried in an oven at 80°C. In this way, the titania layer-formed glass substrate of Test Example 1 and the titania-silica composite oxide layer-formed glass substrates of Test Examples 2 to 4 were prepared.

[0033]

[0034] (Test Examples 5 to 8) <Preparation of Alumina and Alumina-Silica Composite Oxide Layer-Formed Glass Substrates> EtOH, an EtOH solution of ALIP, a TEOS hydrolyzate, a 0.1 M HCl solution, and MilliQ water were added to a glass bottle in the proportions shown in Table 2 and stirred at 300 rpm for 20 hours. The reaction solution was transferred to a polypropylene staining tray (Fisher Scientific), and the same glass substrates used in Test Examples 1 to 4 were immersed in the solution for 1 hour. The glass substrates were then removed and placed in a 50°C oven for 1 hour. The glass surfaces were then washed with MilliQ water and dried in an 80°C oven. In this manner, the alumina layer-formed glass substrate of Test Example 5 and the alumina-silica composite oxide layer-formed glass substrates of Test Examples 6 to 8 were prepared.

[0035]

[0036] <Preparation of Glass Substrates with Titania-Alumina Composite Oxide Layers> EtOH, TTIP, ALIP, 0.1 M HCl solution, and Milli-Q water were added to a glass bottle in the proportions shown in Table 3 and stirred at 300 rpm for 20 hours. The reaction solution was transferred to a polypropylene staining tray (Fisher Scientific), and the same glass substrates used in Test Examples 1 to 4 were immersed in the solution for 1 hour. The glass substrates were then removed and placed in a 50°C oven for 1 hour. The glass surfaces were then washed with Milli-Q water and dried in an 80°C oven. In this manner, the titania-alumina composite oxide layer-formed glass substrates of Test Examples 9 to 12 were prepared.

[0037]

[0038] - Measurement of isoelectric point - The isoelectric points of the surface-modified glass substrates of Test Examples 1 to 12 were measured. The surface charge of these surface-modified glass substrates changes depending on the pH, and the isoelectric point is the pH at which the surface charge becomes zero. Particles charged with an opposite polarity to the surface charge of the substrate are electrostatically adsorbed to the substrate. Therefore, the isoelectric point was determined from the presence or absence of adsorption of silica colloid particles to the substrate. HCl and NaOH dispersions of different concentrations containing 0.1 vol% silica colloid particles were prepared and dropped onto the prepared glass substrates. The presence or absence of particle adsorption was observed using an inverted optical microscope (ECLIPSE, Ti-S, Nikon). The results are shown below.

[0039] (Isoelectric Point of Titania-Silica Composite Oxide-Modified Glass Substrate) The surface-modified glass substrates of Test Examples 1 to 4 were observed for the presence or absence of adsorption of silica colloidal particles to the glass substrate. As shown in Figure 4, the minimum HCl concentration at which silica colloidal particles adsorbed to the substrate was 40 μM for the surface-modified glass substrate of Test Example 1, 50 μM for the surface-modified glass substrate of Test Example 2, 200 μM for the surface-modified glass substrate of Test Example 3, and 600 μM for the surface-modified glass substrate of Test Example 4. These results demonstrate that the isoelectric point of the surface-modified glass substrate can be controlled by changing the ratio of TTIP to TEOS.

[0040] Furthermore, for the titania-silica composite oxide-modified glass substrates of Test Examples 1 to 4, the pH values ​​corresponding to the measurement results of the isoelectric points of the surface-modified glass substrates were calculated and are shown in Figure 5. This graph demonstrates that the isoelectric point of the surface charge can be controlled by changing the mixing ratio of TTIP and TEOS. Note that in Test Example 1, in which TEOS was not mixed, the isoelectric point was lower than the predicted isoelectric point of 6 for titania because there was a portion of the glass substrate surface that was not surface-modified with TTIP.

[0041] (Isoelectric Point of Silica-Alumina Composite Oxide-Modified Glass Substrate) The surface-modified glass substrates of Test Examples 5 to 8 were observed for the presence or absence of adsorption of silica colloidal particles to the glass substrate. As shown in Figure 6, the minimum concentration at which silica colloidal particles adsorbed to the substrate was 600 µM NaOH for the surface-modified glass substrates of Test Examples 5 and 6, 100 µM NaOH for the surface-modified glass substrate of Test Example 7, and 550 µM HCl for the surface-modified glass substrate of Test Example 8. These results demonstrate that the isoelectric point of the surface-modified glass substrate can be controlled over a wide range by changing the ratio of ALIP to TEOS.

[0042] 7 shows the pH values ​​corresponding to the isoelectric point measurements of the surface-modified glass substrates of Test Examples 5 to 8. This graph demonstrates that the isoelectric point of the surface charge can be controlled by changing the mixture ratio of ALIP and TEOS.

[0043] Relationship between the baking temperature and isoelectric point of TTIP-modified glass substrates. The relationship between the baking temperature and isoelectric point of TTIP-modified glass substrates was investigated. 40 mL of ethanol, 186 μL of 0.1 M HCl, 8 μL of Milli-Q water, and 40 μL of TTIP were added to a glass bottle and stirred at 300 rpm for 20 hours at room temperature. The reaction solution was transferred to a polypropylene staining tray, and an optical microscope cover glass was immersed in the glass substrate for 1 hour. The glass substrate was then removed and placed in a 50°C oven for 1 hour. It was then placed in a baking furnace and baked at 200, 350, and 500°C for 30 minutes. The glass substrate surface was then removed from the oven, washed with Milli-Q water, and dried in an 80°C oven. The isoelectric points of the resulting baked TTIP-modified glass substrates were measured for the presence or absence of silica colloidal particle adsorption.

[0044] As a result, as shown in Figure 8, it was found that silica particles were adsorbed to the glass substrates baked at 200°C and 350°C at HCl concentrations of 100 μM or more, and that silica particles were adsorbed to the glass substrates baked at 500°C at HCl concentrations of 200 μM or more. In other words, it was found that the isoelectric point shifts to the acidic side at a baking temperature of 500°C.

[0045] (Isoelectric Point of Titania-Alumina Composite Oxide-Modified Glass Substrate) The surface-modified glass substrates of Test Examples 9 to 12 were observed for the presence or absence of adsorption of silica colloidal particles to the glass substrate. As shown in FIG. 9 , the minimum concentration at which silica colloidal particles adsorbed to the substrate was 20 μM HCl for the surface-modified glass substrate of Test Example 9, 20 μM HCl for the surface-modified glass substrate of Test Example 10, no additive for the surface-modified glass substrate of Test Example 11, and 100 μM NaOH for the surface-modified glass substrate of Test Example 12. These results demonstrate that the isoelectric point of the surface-modified glass substrate can be controlled by changing the ratio of TTIP to ALIP.

[0046] 10 shows the pH values ​​corresponding to the isoelectric point measurements of the surface-modified glass substrates of titania-alumina composite oxide-modified glass substrates of Test Examples 9 to 12. This graph demonstrates that the isoelectric point of the surface charge can be controlled by pH in accordance with the change in the mixing ratio of TTIP and ALIP.

[0047] <Preparation of MIM-Type SPR Measurement Substrate> (Example 1) Substrate Preparation Step S1: Gold-deposited glass (22 mm × 26 mm × 0.55 mm, gold-deposited film thickness 30 nm) was prepared and immersed in Scat® 20X-N (Dai-ichi Kogyo Seiyaku Co., Ltd.) for one day and then washed with Milli-Q water. The front and back surfaces of the gold-deposited glass were then treated for 10 minutes each using a UV ozone cleaning and modification device. The glass was then immersed in concentrated sulfuric acid for at least 24 hours and thoroughly washed with Milli-Q water. The washed gold-deposited glass was then immersed in a solution of 3-mercaptopropyltrimethoxysilane (MEPTMS, Tokyo Chemical Industry Co., Ltd.) in 40 mM aqueous methanol (MeOH) for 3 hours, followed by immersion in 0.01 M NaOH for 2 hours to introduce methoxysilane groups onto the gold surface.

[0048] A metal oxide layer was then formed on the gold-deposited glass substrate, whose gold surface was chemically modified with methoxysilane groups, using the following method. Aluminum isopropoxide (ALIP) was dissolved in ethanol to prepare a 0.097 wt% ALIP solution. In a separate glass bottle, 18 mL of the ALIP solution, 21.8 mL of EtOH, 0.020 mL of titanium tetraisopropoxide (TTIP) solution, 0.186 mL of 0.1 M HCl, and 0.008 mL of ultrapure water were added and stirred at room temperature for 20 hours. The TTIP:ALIP molar ratio was 5:5. The gold-deposited glass substrate, whose gold surface was chemically modified with methoxysilane groups, was immersed in the stirred mixture and shaken for 1 hour, then removed and kept at 50°C for 1 hour. The substrate surface was then rinsed with Milli-Q water and dried at 80°C. In this way, a substrate was produced in which a titania-alumina composite oxide coating with a Ti:Al molar ratio of 5:5 was laminated on gold-deposited glass. The isoelectric point of this titania-alumina composite oxide coating was measured in the same manner as in Tests 1 to 12 described above, and was found to be 7.0.

[0049] Colloidal Crystal Dispersion Preparation Step S2: AU Colloid Solution-SC (Tanaka Kikinzoku Kogyo Co., Ltd.) was used as gold colloid particles. Because unmodified gold colloid particles have a low surface charge, charged colloidal crystals are difficult to form. Therefore, negatively charged carboxyl groups were introduced using 11-mercaptoundecanoic acid (MUA, Sigma-Aldrich, St. Louis, Missouri, USA) in the following manner. A centrifuge tube was charged with 30 ml of ultrapure water, 0.1 ml of 1 M NaOH, and 10 ml of Au particle dispersion (approximately 0.02 wt%). Approximately 0.00896 g of MUA was added, and the mixture was shaken for 24 hours to introduce carboxyl groups onto the particle surface. The resulting gold particles were sedimented by centrifugation (1500 rpm, 20 min). After removing the supernatant, fresh ultrapure water was added, and the particles were washed by ultrasonic dispersion and centrifuging three times. The resulting dispersion was centrifuged (1500 rpm, 20 min) to remove the supernatant, and 30 ml of ultrapure water and 0.02 ml of 0.01 M NaOH were added. The mixture was then centrifuged (1200 rpm, 30 min) to precipitate the suspension. The pH during precipitation was calculated as follows: (1) Type of dissolved ions: The cation was H + and Na + , the anion is OH - Only. (2) Na + Ion concentration: [Na+] = 0.01 × 0.02 / (30+0.02) = 6.66× 10 -6 M (3) Electroneutrality condition [H + ]+[Na + ] = [OH - ], i.e., [H + ]=[OH - ] - [Na + ] (4) [H + ] and [OH - ] ionic product [H + ] [OH - ] = Kw= 10 -14 From the above, [H + ]=[OH - ] - [Na +] = Kw / [H + ] - [Na + ] [H + ] 2 = -[Na + ][H + ] + Kw , [H + ] 2 +[Na + ][H + ] - Kw =0 From this equation, [H + ] = 1.5 x 10 -9 The pH was 8.8. The supernatant was removed, ion exchange resin was added, and the solution was left to stand for 1 hour. Observation under an optical microscope revealed that the gold particles had formed colloidal crystals.

[0050] Transfer Step S3: The gold colloid dispersion prepared in the colloidal crystal dispersion preparation step S2 was concentrated, and salts and carbon dioxide were removed using an ion exchange resin. The purified gold colloid dispersion was then dropped onto the surface of the substrate prepared in the substrate preparation step S1. After leaving it to stand for 10 minutes, it was washed with ultrapure water and stored in ultrapure water. During storage in ultrapure water, the gold colloid dispersion absorbed carbon dioxide from the air, causing the pH to change across the isoelectric point. The mechanism is as follows: (1) CO2 in the atmosphere dissolves in the water as CO2 (gas). (2) The CO2 (gas) dissolved in the water reacts with water to produce carbon dioxide: CO2 + H2O → H2CO3. (3) Carbonic acid dissociates to produce H+, decreasing the pH: H2CO 3 → H++HCO3 - The concentration of carbon dioxide in the atmosphere is 0.04%, and the equilibrium concentration in water is calculated using Henry's law to be 1.4 x 10 -5 M is calculated. H2CO3 → H + + HCO3 - The acid dissociation constant of at 25°C is Ka = 4.4 × 10 -7 From this value, [H ＋ ] = 2.8 × 10 -6M, which is calculated to be pH 5.6. On the other hand, the isoelectric point of the titania-alumina composite oxide coating prepared in Example 1 is 7.0. Therefore, when the substrate of Example 1 is left in an air atmosphere and carbon dioxide gas in the air is dissolved, the pH near the two-dimensional gold colloidal crystals changes to 5.6, crossing the isoelectric point (7.0) of the substrate.

[0051] Example 2 In Example 2, 3-mercapto-1-propanesulfonate (MPS, Sigma-Aldrich, St. Louis, Missouri, USA) was used as a surface modifier for gold colloid particles, and carboxyl groups were introduced using the following method. To 30 mL of Au particle dispersion (approximately 0.007 wt%), 1.7 mL of 1 mM aqueous solution of sodium polystyrene sulfonate (NaPSS) (average molecular weight 70,000) was added to prevent aggregation, and the mixture was allowed to stand at 30°C for 3 hours. The mixture was then centrifuged, the supernatant removed, and ultrapure water was added to bring the total volume to 5 mL. 5 mL of 2 mM MPS aqueous solution was added to the gold dispersion, and the mixture was sonicated for 5 minutes to homogenize the particles, introducing sulfonate groups onto the particle surface. The resulting gold particles were then sedimented by centrifugation (1500 rpm, 20 min). After removing the supernatant, the particles were washed by adding fresh ultrapure water, ultrasonically dispersing, and centrifuging again, three times. The molar ratio of titania to alumina in the titania-alumina composite oxide coating was 7:3. The isoelectric point of this titania-alumina composite oxide coating was measured using the same method as in Tests 1 to 12 described above, and was found to be 4.7. The remaining aspects were the same as in Example 1, and detailed explanations will be omitted. An electron microscope photograph of the two-dimensional colloidal crystals thus obtained on the surface of the substrate of Example 2 is shown in Figure 11.

[0052] Comparative Example 1 In Comparative Example 1, an MIM-type SPR measurement substrate was fabricated using the following method. Substrate Preparation Step S1: Substrate preparation step S1 was performed using the method shown in Figure 13. Gold-deposited glass (22 mm x 26 mm x 0.55 mm, gold-deposited film thickness 30 nm, Kenis Co., Ltd.) was prepared and immersed in Scat® 20X-N (Dai-ichi Kogyo Seiyaku Co., Ltd.) for one day and then washed with Milli-Q water. The front and back surfaces of the gold-deposited glass were then treated for 10 minutes each using a UV ozone cleaning and modification device. The glass was then immersed in concentrated sulfuric acid for at least 24 hours and thoroughly washed with Milli-Q water. The washed gold-deposited glass was then immersed for 3 hours in a solution of 3-mercaptopropyltrimethoxysilane (MEPTMS, Shin-Etsu Chemical Co., Ltd.) dissolved in 40 mM aqueous methanol (MeOH) to introduce methoxysilane groups onto the gold surface. The gold-deposited glass was then thoroughly rinsed with MeOH solution and immersed in 0.01 M NaOH solution for 2 hours. This dehydration polymerization of the methoxysilane groups resulted in the surface being coated with silanol groups. Further chemical modification was performed with APTES to introduce an amino-modified silicon oxide film onto the substrate surface. The isoelectric point of this amino-modified silicon oxide film was measured using the same method as in Tests 1-12, and was found to be 8.2.

[0053] Colloidal Crystal Dispersion Preparation Step S2 The gold colloidal crystal dispersion prepared in Comparative Example 1 was the same as that prepared in Example 1 and was prepared by the same procedure.

[0054] Transfer Step S3: The gold dispersion prepared in the colloidal crystal dispersion preparation step S2 was concentrated and purified using an ion exchange resin. The purified dispersion was then dropped onto the surface of the substrate prepared in the substrate preparation step S1, after which the surface was washed with ultrapure water and stored in ultrapure water. Thus, the MIM-type SPR measurement substrate of Comparative Example 1 was obtained. An electron microscope photograph of the two-dimensional colloidal crystals on the surface of the substrate of Comparative Example 1 obtained in this manner is shown in Figure 12. A comparison of Figures 11 and 12 reveals that the crystallinity of the two-dimensional colloidal crystals in Example 2 is higher than that of Comparative Example 1.

[0055] <Evaluation> The MIM type SPR measurement substrates of Examples 1 and 2 and Comparative Example 1 were evaluated by the following test methods.

[0056] (Refractive index measurement using MIM-type SPR measurement substrate) The refractive index of water and an ethylene glycol-water mixed solvent was measured for the MIM-type SPR measurement substrates of Example 1, Example 2, and Comparative Example 1. The MIM-type SPR measurement substrate was placed in a flow cell, and water and an ethylene glycol-water mixed solvent were allowed to flow through, and the reflectance spectrum was measured using a fiber-type spectrophotometer (HR4000, Ocean Optics Co. Ltd., FL, USA). As a result, for the MIM-type SPR measurement substrate of Comparative Example 1, the peak shifts for 100% water, 40% EG aqueous solution, and 80% EG aqueous solution were 720.84 nm, 728.23 nm, and 735.59 nm, respectively, and the peak shift (refractive index unit, RIU) per refractive index = 1 was 179.95 nmRIU. -1 On the other hand, the SPR peak wavelengths of the MIM-type SPR measurement substrate of Example 1 were 721.17 nm, 736.10 nm, and 744.00 nm, respectively, and the peak shift amount per refractive index unit (RIU) = 1 was 279.92 nmRIU. -1 In Example 2, the peak shift amounts were 336.20 nmRIU at 727.12 nm, 742.89 nm, and 754.60 nm, respectively. -1 This is 1.87 times that of Comparative Example 1.

[0057] (SPR Measurement) - SPR Measurement Using Polyelectrolyte Layer-by-Layer Films Polyelectrolyte layer-by-layer films were laminated on the surface of the MIM-type SPR measurement substrates of Example 1 and Comparative Example 1, and SPR measurements were performed. Details are provided below. - Method for Forming Polyelectrolyte Layer-by-Layer Films A 1 mM aqueous solution of sodium polystyrene sulfonate (NaPSS) (average molecular weight 70,000) and a 1 mM aqueous solution of polyallylamine (PAA) (average molecular weight 50,000) were prepared. The MIM-type SPR measurement substrate was placed in a flow cell, and the NaPSS aqueous solution, pure water, PAA aqueous solution, and pure water were flowed in this order. By repeating this process, polyelectrolyte layer-by-layer films with various lamination numbers were formed on the surface of the MIM-type SPR measurement substrates. The thickness of a laminated film consisting of one layer each of NaPSS and PAA can be estimated to be 4 nm (Plasmonics 9, 773-780 (2014). Macromolecules 2004, 37 (19), 7285-7289).

[0058] SPR measurements were performed on the MIM-type SPR measurement substrate of Example 1 on which the polyelectrolyte layer-by-layer film was formed, obtained in this manner. SPR measurements were also performed on an SPR measurement substrate (hereinafter referred to as an LSPR measurement substrate) in which two-dimensional gold colloidal crystals were adsorbed onto a glass substrate without gold deposition, treated in the same manner as in Example 1.

[0059] As a result, it was found that the MIM-type SPR measurement substrate of Example 1 exhibited approximately twice the peak shift compared to the LSPR measurement substrate (see Figure 14). Figure 15 shows the relationship between the SPR peak shift (Δλ) and the number of layers in the MIM-type SPR measurement substrate of Example 1. When either of two types of polymers is layered, the number of layers is set to 1. The graph shows, from left to right, the spectra before polymer addition, when PAA is added, when NaPSS is further added, when a second PAA is further added, and when NaPSS is further added a second time.

[0060] Figure 16 shows the results of SPR measurements of the MIM-type SPR measurement substrates of Example 1 and Comparative Example 1. This shows the spectral change when NaPSS and PAA are alternately laminated, with the lowest wavelength peak and dip observed before the addition of the polymer. It can be seen that the addition of the polymer shifts the spectrum toward longer wavelengths. The relationship between the SPR peak shift (Δλ) and the number of layers, determined from this graph, is shown in Figure 17. These results demonstrate that the MIM-type SPR measurement substrate of Example 1 exhibits a peak shift that is more than twice as large as that of Comparative Example 1.

[0061] ・SPR measurement of MIM-type SPR measurement substrate modified with bovine serum albumin (BSA)

[0062] <Measurement (Part 1)> The globular protein bovine serum albumin (BSA) was immobilized on the surface of the MIM-type SPR measurement substrates of Example 2 and Comparative Example 1 using the amine coupling method, and the responsiveness to protein adsorption was measured. Because the number of surface charges on BSA varies with pH, ​​the amount of binding also varies with pH. Therefore, various buffers (pH buffer solutions) were used to determine the optimal pH conditions for binding with BSA (see Measurements (Parts 2) and (Parts 3) below). The buffers used are shown in Table 4. To prevent nonspecific binding to groups other than the carboxyl group of BSA, the surfactant polyoxyethylene (20) sorbitan monolaurate was added to the buffer. This covered the gold surface with the surfactant, preventing adsorption to the gold surface of groups other than the carboxyl group.

[0063]

[0064] The liquid was delivered to the substrate through a fluororesin tube using a peristaltic pump, and the reflection spectrum was measured to determine the SPR dip wavelength. First, the flow path was washed with Buffer 1 (flow rate 0.64 ml / min), and the reflection spectrum (1) was measured. Next, 0.020 ml of a mixed solution of EDC (200 mM) and NHS (50 mM) dissolved in ultrapure water was injected three times (flow rate 0.064 ml / min). Six minutes after the initial injection, the reflection spectrum (2) was measured. The flow path was washed with Buffer 2 (in Example 2, Buffer 1 was the same as Buffer 2) (flow rate 0.64 ml / min), and the reflection spectrum (3) was measured. Finally, 0.020 ml of BSA (0.3 mg / ml, dissolved in Buffer 2) was injected three times (flow rate 0.064 ml / min). After 400 seconds, the reflection spectrum (4) was measured. The dip wavelength and the shift amount Δλ in each process when the MIM-type SPR measurement substrates of Example 2 and Comparative Example 1 were used are shown in the upper part of Table 4. When BSA was added, no clear shift was observed in the MIM-type SPR measurement substrate of Comparative Example 1, but a clear shift of 4.6 nm was observed in the MIM-type SPR measurement substrate of Example 2.

[0065] <Measurement (Part 2)> Measurement (Part 2) used the buffer shown in the middle of Table 4. The rest was the same as Measurement (Part 1). When BSA was added, a clear shift of several nm was observed in both the MIM-type SPR measurement substrates of Example 2 and Comparative Example 1, but the MIM-type SPR measurement substrate of Example 2 had a larger shift and better sensitivity.

[0066] <Measurement (Part 3)> Measurement (Part 3) used the buffer shown in the lower part of Table 4. Other conditions were the same as Measurements (Part 1) and (Part 2). As a result, with the MIM-type SPR measurement substrate of Comparative Example 1, there was almost no change even when BSA was added, whereas with the MIM-type SPR measurement substrate of Example 2, a shift of approximately 1 nm was observed.

[0067] Example 3 The MIM-type SPR measurement substrate of Example 3 is a substrate intended for measuring proteins such as BSA, and was prepared by introducing carboxyl groups onto the surface of the MIM-type SPR measurement substrate of Example 2 using an aqueous silane coupling agent (X-12-1135, manufactured by Shin-Etsu Silicones Co., Ltd.) having a carboxylic acid group as an organic functional group. The reason for introducing the carboxyl groups was that it was thought that the sensitivity of SPR measurements would be increased by the adsorption of proteins such as BSA to the carboxyl groups.

[0068] Example 4 The MIM-type SPR measurement substrate of Example 4 is also a substrate for measuring proteins such as BSA, and was prepared by modifying the surface of the insulator layer of the MIM-type SPR measurement substrate of Example 2 with APTES, and then amide-bonding carboxymethyldextran (CMD) using the amine coupling method.

[0069] (Example 5) The MIM-type SPR measurement substrate of Example 5 is also a substrate for measuring proteins such as BSA, and was prepared by modifying the surface of the insulator layer of the MIM-type SPR measurement substrate of Example 2 with 3-methacryloxypropyltrimethoxysilane, followed by polymerization of acrylic acid monomers in a methanol solution to introduce polyacrylic acid.

[0070] The SPR peak shift due to BSA adsorption was measured using the MIM-type SPR measurement substrates of Examples 3 to 5. Measurements were performed using sodium acetate buffer (pH 5.0) with a BSA concentration of 300 μg / mL. SPR measurements were performed using the same method as described in <Measurement (Part 1)>. The SPR peak shift was determined based on the dip wavelength change from 650 to 750 nm. As shown in Table 5, the peak shift due to BSA adsorption was clearly confirmed in all of Examples 3 to 5. However, when measurements were performed in HEPES buffer (pH 7.4), the peak shift value fluctuated over time, making measurement difficult. This was presumably due to hydrolysis of the metal oxide layer.

[0071] Example 6: The MIM-type SPR measurement substrate of Example 6 was the same as that of Example 2, except that the surface of the metal oxide layer was treated with tetraethylsiloxane (TEOS). Specifically, the substrate was immersed for two hours in a solution of 10 ml of 1 vol% TEOS solution (EtOH:water = 90:10) and 0.285 ml of 28% aqueous ammonia, forming a TEOS hydrolysate layer on the metal oxide layer. The same process as in Example 3 was then carried out.

[0072] The SPR peak shift due to BSA adsorption was measured using the MIM-type SPR measurement substrate of Example 6. Measurements were performed in HEPES buffer (pH 7.4) using the same method as in Examples 3 to 5. As a result, as shown in Table 6, a peak shift (10.65 nm) due to BSA adsorption was clearly observed, with little fluctuation over time. This result is thought to be due to the fact that hydrolysis of the metal oxide layer was prevented by treating the surface of the metal oxide layer with tetraethylsiloxane (TEOS).

[0073] <Preparation and Evaluation of Immunosensor> Immunosensors were prepared by chemically modifying the surface of the MIM-type SPR measurement substrate of Examples 1 and 2 with an anti-GST antibody, and their characteristics were measured.

[0074] Example 7 The immunosensor of Example 7 was fabricated as follows. The MIM-type SPR measurement substrate of Example 1 was placed in a flow cell (cell size: diameter 3 mm, height 3 mm, buffer replacement operation 0.64 mL / min, reaction solution flow rate 0.16 mL / min, antibody flow rate 0.064 mL / min). Then, using a Helistar pump, a running buffer (HBS-EP+ manufactured by Cytiva) was passed through the flow channel for cleaning. Next, 60 μL of a 1:1 mixture of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC, 400 μM) and N-hydroxysuccinimide (NHS, 100 μM) aqueous solutions was passed through the flow channel for 6 minutes to activate the carboxyl groups. After washing the flow channel with sodium acetate buffer (10 mM Na-Ac, pH 5.0), 60 μL of sodium acetate buffer (10 mM Na-Ac, pH 5.0) containing an anti-GST antibody was passed through the flow cell for 10 min. The antibody was immobilized by amide bonding with the amino group of the anti-GST antibody, resulting in the immunosensor of Example 7. The antibody was passed through the flow cell at a concentration of 300 μg / mL, and reflectance measurements were performed using a fiber spectrophotometer. An SPR peak shift of approximately 1.7 nm was observed. Subsequently, 60 μL of antigen (GST 20 μg / mL) was passed through the flow cell, but no clear shift in the SPR peak was observed.

[0075] Example 8 The immunosensor of Example 8 was fabricated by the following method. A carboxyl group was introduced by bonding a silane coupling agent (X-12-1135, manufactured by Shin-Etsu Silicones) having a carboxyl group to the metal oxide layer on the surface of the MIM-type SPR measurement substrate of Example 2. Then, as in the case of the MIM-type SPR measurement substrate of Example 7, an anti-GST antibody was bonded in a sodium acetate buffer solution (10 mM Na-Ac, pH 5.0) to produce the immunosensor of Example 8. The antibody was flowed through a flow cell at a concentration of 300 μg / mL, and reflectance measurements were performed using a fiber spectrophotometer. An SPR peak shift of approximately 15.7 nm was observed. Furthermore, the antigen was flowed through a flow cell at a concentration of 20 μg / mL, and reflectance measurements were performed using a fiber spectrophotometer. An SPR peak shift of approximately 1.0 nm was observed.

[0076] Example 9 In Examples 7 and 8, the SPR peak shift was successfully observed in sodium acetate buffer (10 mM Na-Ac, pH 5.0). However, measurements in HEPES buffer (pH 7.4) exhibited time-varying shift values, making measurement difficult. Therefore, in the immunosensor of Example 9, the MIM-type SPR measurement substrate of Example 6, in which the surface of the metal oxide layer was treated with tetraethylsiloxane (TEOS), was used to prevent surface hydrolysis and thereby prevent time-varying peak shift values. Specifically, in the immunosensor of Example 9, the MIM-type SPR measurement substrate of Example 6 was prepared, and anti-GST antibody was bound to it using the same procedure as for the immunosensor of Example 8. Then, the antibody was flowed through a flow cell at a concentration of 300 μg / mL in sodium acetate buffer (10 mM Na-Ac, pH 5.0), and reflectance was measured using a fiber spectrophotometer. As a result, an SPR peak shift of 5.0 nm was observed. Furthermore, when an antigen concentration of 20 μg / mL was flowed into a flow cell in HEPES buffer (pH 7.4) and the reflectance was measured using a fiber spectrophotometer, an SPR shift of approximately 0.53 nm was observed (see Table 7). The pH of body fluids is approximately 7.4, and the immunosensor of Example 9 was found to be suitable for use as a biosensor because it can be used within this range.

[0077] <FDTD Simulation (Part 1)> To investigate the optimal structure of the MIM-type SPR measurement substrate, we performed computer simulations using the finite-difference time-domain (FDTD) method. The calculations were performed using commercially available software (OptiFDTD). An example of a calculation model for the MIM structure is shown in Figure 18. Here, the gold particle diameter is 150 nm, the dielectric layer thickness is 6 nm, the dielectric refractive index is 1.52, and the gold deposition layer thickness is 30 nm. The incident light is a sine-modulated Gaussian pulse with a wavelength of 0.55 μm. Furthermore, a non-uniform mesh was introduced for the region with a significant refractive index change near the gold material. For comparison with experiments, simulations were performed under three medium conditions: water (refractive index 1.333), a 40% EG aqueous solution (refractive index 1.3759), and an 80% EG aqueous solution (refractive index 1.4149), and the RIU of each dip was calculated. When multiple dips were observed, the one with the greatest change was used for comparison.

[0078] We then performed a simulation to examine the interparticle distance dependence. The results are shown in Figure 19. The shift tended to increase with increasing interparticle distance. At an interparticle distance of 560 nm, a shift of 516.98 nm RIU-1 was observed, indicating a sensitivity 2.87 times higher than that of Comparative Example 1. However, a significant decrease in the shift was observed between interparticle distances of 450 nm and 470 nm. Figure 20 shows a comparison of the simulation results with the experimental values. This figure confirms that the behavioral trends between the experimental and simulation results are similar. In the experiment, 0.003 mL of approximately 0.01 mL of gold particle dispersion was dropped, and 0.003 mL of ultrapure water was added to the remaining dispersion, followed by another 0.003 mL drop. Substrates with three levels of interparticle distance were fabricated using the above method, and SPR measurements were performed. Further dilution and subsequent dropping did not result in an increase in the interparticle distance. The average interparticle distance was determined from microscopic images.

[0079] Figure 21 shows the results of an FDTD simulation of the effect of the thickness of the gold vapor deposition layer. The interparticle distance was assumed to be 560 nm. A thickness of 20 nm was the best, showing a shift of RIU = 585.41 nm. The thickness of the dielectric layer was also investigated by FDTD simulation with an interparticle distance of 560 nm and gold vapor deposition layer thicknesses of 20 nm and 30 nm. When the gold vapor deposition layer thickness was 20 nm, the sensitivity was 585 nmRIU when the dielectric layer thickness was 6 nm and 10 nm. -1 and 621 nmRIU -1 The shift increased with increasing dielectric layer thickness, but the SPR dip disappeared when the thickness was 20 nm. When the gold deposition layer thickness was 30 nm, the sensitivity was 517 nmRIU for dielectric layer thicknesses of 6 nm, 7 nm, and 8 nm. -1 ,526 nmRIU -1 , 621 nmRIU -1 It was.

[0080] The above simulations assumed that the MIM-type SPR measurement substrate was placed in a medium with a uniform dielectric constant (refractive index). However, when used as an immunosensor, organic molecules such as protein molecules adhere only to the flat surfaces of the gold particles and metal oxide layer, while the rest of the surface is solvent, resulting in a non-uniform dielectric constant (refractive index). FDTD simulations were performed for this case. Figure 22 shows the case where gold particles are chemically modified with antibodies to adsorb the antigen to be measured. The "adsorption layer" is shown schematically with diagonal lines. Figure 23 shows the results assuming a protein with a dielectric constant of 1.9 as the adsorbed molecule. The thickness is varied from left to right: 8, 16, 20, 24, 40, 48, 52, 60, and 68 nm. As the thickness increases, the gold particle diameter is set to 150 nm and the dielectric layer thickness is set to 6 nm.

[0081] Figure 24 shows the case where an antibody is chemically modified on the surface of a metal oxide layer, allowing the antigen to be adsorbed onto the antibody. The "adsorption layer" is shown with a diagonal line. Figure 25 shows the results assuming a protein with a dielectric constant of 1.9 as the adsorbed molecule. The interparticle distance is 330 nm, and the thickness of the adsorption layer is 7 nm. Calculations were performed for various values ​​of θ, where θ is the volume fraction of the molecules in the adsorption layer. θ = 1 for 100% adsorption, and θ = 0 for no adsorption. The diameter of the gold particles was 150 nm, and the thickness of the dielectric layer was 6 nm. From the left to right, the graphs are for θ = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0.

[0082] Figure 26 shows the results when the adsorbed molecule is assumed to be a protein with a dielectric constant of 1.9. The interparticle distance is 560 nm, and the thickness of the adsorbed layer is 7 nm. Calculations were performed for various values ​​of θ. From the left graph, the values ​​are θ = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0.

[0083] Simulations were performed to compare the results of chemically modifying the surface of a metal oxide layer with an antibody and the results of chemically modifying the surface of gold particles with an antibody. The results showed that the dip shift Δλ varied depending on the distance between gold particles, and which method was more effective (see Table 8). In practice, gold colloid particles with a particle size of approximately 150 nm can be used, and the interparticle distance can be easily adjusted to approximately 300-400 nm. Therefore, the method of adsorbing antibodies onto the surface of a metal oxide layer (see Figure 24) resulted in a larger wavelength shift.

[0084]

[0085] <FDTD Simulation (Part 2)> The effect of the gold particle distance on the plasmon peak was simulated using the FDTD method. The gold particle diameter was 150 nm, the dielectric layer thickness was 6 nm, the dielectric refractive index was 1.52, and the gold deposition layer thickness was 30 nm. Figure 27 shows the calculated results of the reflectance spectra for MIM structures with three interparticle distances d. These results indicate that the dip position varies depending on the interparticle distance. Furthermore, in Figure 19, which shows the simulation results for interparticle distance dependence, the decrease in the shift between 450 and 470 nm is understood to be due to the dip position disappearing from the visible light range.

[0086] The present invention is not limited to the above-described embodiments and examples, and various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention.

[0087] The MIM-type SPR measurement substrate of the present invention has a two-dimensional colloidal crystal on an insulating film, in which metal colloid particles are regularly arranged at a fixed distance, and the crystal structure has few defects. This makes it possible to construct a localized surface plasmon sensor with high detection sensitivity. Furthermore, it does not require complex pattern formation technology to control the arrangement of the metal particles, making it easy to manufacture and enabling significant cost reductions through mass production.

[0088] S1...substrate preparation step, S2...colloidal crystal dispersion preparation step, S3...transfer step 1...substrate, 2...metal film, 3...metal film-coated substrate, 4...metal oxide layer, 5...metal substrate with insulating film, 6...three-dimensional charged colloidal crystal dispersion, 7...two-dimensional charged metal colloidal crystal, 8...MIM-type SPR measurement substrate,

Claims

1. A method for manufacturing an MIM-type SPR measurement substrate, comprising: a substrate preparation step of preparing a substrate having a metal oxide layer having an isoelectric point of 4 or more and 8 or less formed on a metal film; a colloidal crystal dispersion preparation step of preparing a three-dimensional charged colloidal crystal dispersion in which charged colloidal crystals made of metal particles are dispersed in a dispersion medium, and the surface charge of the charged colloidal crystals has the same sign as the surface charge of the metal oxide layer; and a transfer step of contacting the metal oxide layer with the charged colloidal crystal dispersion, and then adjusting the surface charge of the metal oxide layer so that it has a different sign from the surface charge of the three-dimensional charged colloidal crystals, and transferring two-dimensional charged metal colloidal crystals onto the metal oxide layer.

2. A method for manufacturing an MIM-type SPR measurement substrate according to claim 1, wherein the change in pH of the three-dimensional charged colloidal crystal dispersion during the transfer step is 4 or less, and the salt concentration is 150 μM or less.

3. A method for manufacturing an MIM-type SPR measurement substrate according to claim 1 or 2, wherein the isoelectric point of the metal oxide layer is 4.5 or more and 7.5 or less.

4. A method for manufacturing an MIM-type SPR measurement substrate according to claim 1 or claim 2, wherein the metal oxide layer is made of a metal oxide, and the metal element in the metal oxide is one of Ti or Al, or at least two of Ti, Si, and Al.

5. A method for manufacturing an MIM type SPR measurement substrate according to claim 1 or 2, wherein the metal oxide layer is made of titania or a titania-containing composite oxide.

6. A method for producing an MIM-type SPR measurement substrate according to claim 1 or 2, wherein the substrate preparation step is carried out by polycondensation of a hydrolyzate of a metal alkoxide-containing solution.

7. A method for manufacturing an MIM type SPR measurement substrate according to claim 1 or claim 2, wherein the metal particles are gold particles.

8. A method for manufacturing an MIM-type SPR measurement substrate according to claim 1 or 2, wherein the coefficient of variation of the particle diameter of the metal particles constituting the three-dimensional charged colloidal crystal is 20% or less.

9. A method for manufacturing an MIM-type SPR measurement substrate according to claim 1 or 2, characterized in that the metal particles constituting the three-dimensional charged colloidal crystal have an average particle diameter of 50 nm or more and 500 nm or less.

10. A method for manufacturing an MIM type SPR measurement substrate according to claim 1 or 2, characterized in that the metal film has a film thickness of 1 nm or more and 100 nm or less.

11. An MIM-type SPR measurement substrate in which a metal oxide layer having an isoelectric point of 4 or more and 8 or less is formed on a metal film, and a two-dimensional colloidal crystal layer made of metal particles is formed on the metal oxide layer.

12. The MIM type SPR measurement substrate according to claim 11, wherein the isoelectric point of the metal oxide layer is 4.5 or more and 7.5 or less.

13. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the metal element in the metal oxide layer is one of Ti or Al, or at least two of Ti, Si and Al.

14. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the metal oxide layer is made of titania or a titania-containing composite oxide.

15. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the metal oxide layer is made of a hydrolysis polycondensate of a metal alkoxide.

16. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the two-dimensional colloidal crystal layer is made of gold particles.

17. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the coefficient of variation of the particle diameter of the metal particles is 20% or less.

18. The MIM type SPR measurement substrate according to claim 11 or 12, wherein the metal particles have an average particle diameter of 50 nm or more and 500 nm or less.

19. An immunosensor in which a metal oxide layer having an isoelectric point of 4 or more and 8 or less is formed on a metal film, a two-dimensional colloidal crystal layer made of metal particles is formed on the metal oxide layer, and an antibody or antigen is bound to the metal oxide layer and / or the metal particles.

20. An immunosensor comprising: a metal oxide layer having an isoelectric point of 4 or more and 8 or less formed on a metal film; a two-dimensional colloidal crystal layer made of metal particles formed on the metal oxide layer; a tetraalkoxysilane hydrolysate layer formed on the surface of the metal oxide layer; and an antibody or antigen bound to the tetraalkoxysilane hydrolysate layer and / or the metal particles.

Images