JPH1194734A - Sensor element, its manufacture, and sensor apparatus using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sensor element utilizing a surface plasmon resonance(SPR) of good characteristics, by forming on a light-transmitting supporting body a mixed layer containing both a metal having free electrons and an inorganic or organic substance. SOLUTION: A sensor element 1 utilizing the SPR has a light-transmitting supporting body 2, and a mixed layer 3 formed on the supporting body 2 and containing both a metal having free electrons and an inorganic or organic substance. For instance, the mixed layer 3 has a gradient composition with a content of the metal reduced in proportion to a distance from the supporting body 2. A part of a depth α1 of the mixed layer 3 from an interface 4 to the supporting body 2 having a large content of metal functions substantially as a metallic thin film. On the other hand, a part of a depth β1 of the mixed layer 3 from an upper face 21 functions substantially as a protecting layer or intermediate layer. When a substance to be tested comes in touch with the upper face 21, a distance from the face 21 to the metallic thin film is practically shortened, with the same result as when the substance to be tested is brought close to the metallic thin film. Accordingly measurement sensitivity is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor element using surface plasmon resonance (SPR), a method for manufacturing the same, and a sensor device using the same.

2. Description of the Related Art Recently, a sensor device called a surface plasmon resonance (SPR) sensor has been proposed (for example, Japanese Patent Laid-Open Publication No. Hei 4-50).
No. 1605, Japanese Translation of PCT International Publication No. Hei 3-502604, Protein Nucleic Acid Enzyme Vol. 37, No. 15 (1992), etc.). This SPR sensor is composed of an evanescent wave generated by total reflection of light on the surface of the metal thin film and a surface plasmon (SP) generated on the surface of the metal film.
This is a sensor that utilizes resonance with the sensor.

[0003] The principle of this sensor is briefly described as follows. If there is light traveling through a layer having a high refractive index, for example, a glass layer, and its incident angle is such that it is totally reflected at an interface with the outside, the light is reflected without exiting the glass. Here, when a metal thin film is brought into contact with the reflecting surface,
A phenomenon is observed in which the light incident at a certain angle among the lights incident so as to be totally reflected is absorbed, that is, the intensity of the reflected light at a certain angle decreases. Such a phenomenon is caused by resonance of the following two waves. First, when light is totally reflected at an interface, a very small wave called an evanescent wave that propagates only on the surface oozes at the interface. On the other hand, a surface wave called surface plasmon may be generated on a thin metal film. When the wavelengths of these two surface waves coincide, resonance occurs, and a part of the light energy is converted to SP.
It is used to excite R, reducing the amount returned as reflected light. At this time, the resonance point between the evanescent wave and the SP of the metal thin film changes depending on the incident angle of light and the refractive index of the medium in contact with the metal thin film. The change in the refractive index of the medium can be known as a phenomenon of the intensity of the reflected light at a certain angle. Since the refractive index of the medium changes according to the concentration of the solute contained in the medium, the change in the concentration of the solute in the medium that comes into contact with the metal thin film can be known.

Further, when a substance having an affinity for a specific target substance is carried on a metal thin film and this target substance is captured near the surface of the metal thin film, this causes a change in the concentration of a solute in a medium in contact with the metal thin film. Has substantially the same meaning as In other words, qualitative and quantitative determination of the target substance becomes possible by using the SPR of the metal surface carrying a substance having an affinity for the target substance.

[0005] The characteristics required for such a sensor using SPR include (a) a sufficiently sharp resonance curve can be obtained, and (b) an evanescent wave and an SP can be measured.
Being in the ripple field of the wave. The above (a) is closely related to the thickness of the metal thin film. In other words, the energy distribution of the evanescent wave exponentially attenuates in the vertical direction from the boundary, so that if the film is thick, the evanescent wave is too weak (in other words, the evanescent wave cannot ooze to the metal surface) and no resonance occurs. . In this sense, the thinner the film, the better. However, if the film is too thin, SP is unlikely to occur.

In general, if the thickness of the metal thin film exceeds about 100 nm, a resonance curve cannot be obtained. Therefore, it is considered that the width of the evanescent wave field is about 100 nm including the metal film from the boundary. Further, the resonance intensity of the two waves becomes maximum when the thickness of the metal thin film is about 50 nm. Therefore, when the thickness of the metal thin film was set to 50 nm, it became clear that the measurement could not be performed unless the test substance was placed within 50 nm from the surface of the metal thin film. However, since the evanescent wave decreases exponentially, the distance between the metal thin film and the test substance needs to be within a very limited range in order to secure a certain level of measurement sensitivity.

Furthermore, in order to prevent corrosion of the metal thin film, it is generally necessary to provide a protective layer so that the test substance such as a solution does not come into direct contact with the metal thin film. Also,
As described above, it is necessary to interpose some kind of intermediate layer in order to stably support the substance capturing the target substance on the metal surface. In order to stably support the substance, the intermediate layer needs to have a certain thickness. However, these protective layers or intermediate layers separate the reaction with the test substance or the target substance from the metal surface, and may lower the measurement sensitivity or cause the measurement to be impossible.

Further, with respect to the above protective film or intermediate layer, the resonance point shifts to a higher incident angle side as the protective layer or the intermediate layer is thicker or the refractive index of the layer is higher. On the other hand, the evanescent wave has such a property that the higher the incident angle is, the narrower its spreading field becomes (the less it spreads out). As a result, the presence of a protective or intermediate layer
It became clear that the measurement sensitivity might be further reduced.

A sensor element using SPR requires a thin metal film from the viewpoint of the evanescent wave field width, while a certain thickness is required from the viewpoint of the resonance intensity of the two waves. In addition, the protective layer or the intermediate layer is required to have a test substance or thinner from the surface of the metal thin film.

As described above, in the sensor element using the SPR, there is a demand that the thickness of the metal thin film and the thicknesses of the protective layer and the intermediate layer contradict each other, and the film thickness or the layer thickness satisfying these requirements is within a very limited range. I will Such a limitation limits the kind of the protective layer or the intermediate layer, the substance supported on the intermediate layer to form the sensitive layer, and further limits the use range of the sensor using the SPR.

[0011]

SUMMARY OF THE INVENTION The present inventors have recently developed a sensor element using SPR by using a mixed layer containing both a metal having free electrons and an inorganic or organic substance.
It has been found that high sensitivity of a sensor element using SPR can be realized. Specifically, by combining the function of a metal thin film and the function of a protective layer or an intermediate layer into one layer, which is a mixed layer, the inconsistent demands required for sensor elements using SPR are taken at a high level. It has been found that the above-mentioned conditions can be satisfied, and more specifically, the influence on the sensitivity of the protective layer or the intermediate layer can be reduced. The present invention is based on this finding.

Therefore, an object of the present invention is to provide a sensor element using SPR having good characteristics.

The sensor element using the SPR according to the present invention comprises a light-transmitting support, (a) a metal having free electrons, and (b) an inorganic or organic substance formed on the support. And a mixed layer containing both.

According to the present invention, the mixed layer has, on one side, substantially the same function as the metal thin film, and on the other side, has the function of a protective layer or an intermediate layer. That is, the mixed layer functions as a metal thin film having a thickness to obtain a good resonance, and at the same time, functions as a protective layer or an intermediate layer to place a reaction with the test substance or the target substance near the metal thin film. As a result, the sensor element according to the invention has a high degree of measurement.

[0015]

The concept of the sensor element is a sensor device used according to the basic principle the present invention of the sensor using the SPR DETAILED DESCRIPTION OF THE INVENTION FIG 1. In FIG. 1, a sensor element 1 according to the present invention comprises a support 2 and a mixed layer 3 provided thereon. The mixed layer 3 has both the function of a metal thin film that causes SPR and the function of a protective film that protects it or an intermediate layer that can form a sensitive layer thereon. Light from a light source 11 is applied to the interface 4 between the support 2 and the mixed layer 3 of the sensor element 1. The prism 5 collects various lights 6 having incident angles θ1 to θ2,
, And exits as reflected light 7. here,
The intensity of light entering at a certain angle of incidence is reduced depending on the refractive index of the substance in contact with the surface 9 on the mixed layer 3 side of the sensor element 1. In the drawing, it is shown that the light indicated by 10 comes out with decreasing intensity. This is read by the measuring means 12.
A calibration curve is prepared in advance for the refractive index of the substance in contact with the surface 9 and the incident angle of light for reducing the intensity, and the refractive index of the substance in contact with the surface 9 can be known based on the calibration curve. In addition,
In this specification, the angle of incidence of light with reduced intensity may be referred to as “resonance angle”.

In the embodiment shown in FIG. 1, light having various incident angles is collected by the prism and reaches the interface 4;
It is of course possible to irradiate the interface 4 with light while gradually changing from 1 to θ2, and to measure which incident angle of the light reduces the intensity. However, from the viewpoint of measurement efficiency, it is clear that it is extremely preferable to use a prism capable of irradiating various kinds of incident light at a time.

Sensor Element The sensor element according to the present invention basically comprises a support and a mixed layer formed thereon. In the present invention, the mixed layer comprises (a) a metal having free electrons;
(B) Both inorganic and organic substances are included. In the present invention, “comprising together” means (a) a metal,
(B) It means that both exist in the mixed layer in a mode in which the function of each of the inorganic substance and the organic substance is exhibited. The mixed layer functions as a metal thin film as described later, and also functions as a protective layer or an intermediate layer.
Therefore, in the present invention, these (a) metals
(B) As long as the inorganic or organic component functions as a metal thin film, a protective layer, or an intermediate layer, the existence form is not limited. A preferred specific example of such a form is a form in which both are physically mixed without being chemically bonded. Further, as another preferable example of the existence form, there is an embodiment in which the particles are partially combined at the contact surface of both particles by ionic bond, covalent bond, or the like.

According to the first preferred embodiment of the present invention, the mixed layer has a gradient composition in which the metal content decreases in proportion to the distance from the support 2. FIG. 2 shows a schematic diagram of the sensor element 1 having such a mixed layer 3. The mixed layer 3 has a large metal content, and a portion at a depth α1 from the interface 4 with the support 2 substantially functions as a metal thin film. On the other hand, a portion of depth β1 from the upper surface 21 of the mixed layer can substantially function as a protective layer or an intermediate layer. The structure of the sensor element when the metal thin film having the thickness α1 and the protective layer or the intermediate layer having the thickness β1 are separately formed on the support 2 is as shown in FIG. Assuming that the test substance comes into contact with the upper surface 21 side of the mixed layer, in the configuration of FIG. 3, the distance to the metal thin film is β1. On the other hand, in the sensor element according to the present invention, if the test substance comes into contact with the upper surface 21 of the mixed layer, the distance from the surface 21 to the metal thin film is β
It is substantially shorter than 1. Therefore, when the sensor element according to the present invention is used, the same effect as when the test substance is substantially brought closer to the metal thin film can be obtained, and the advantage that the measurement sensitivity can be increased can be enjoyed.

According to a preferred embodiment of the present invention, the mixed layer 3
Is preferably 50% or less, more preferably 20% or less.

Further, in addition to the embodiment in which the content of the metal is continuously changed as shown in FIG.
Has a plurality of layers 41 to 47, and the metal content in each layer is constant, but the metal content may be changed between adjacent layers.

In this embodiment, the thickness of the mixed layer 3 may be appropriately determined in consideration of various conditions, but the lower limit is preferably about 10 nm, more preferably about 20 nm, and most preferably about 30 nm. It is. The upper limit is preferably about 100 nm, more preferably about 80 nm, and most preferably about 70 nm.

According to a second aspect of the present invention, the mixed layer may have a substantially homogeneous composition. FIG. 5A shows a sensor element having such a mixed layer 3. In this embodiment, the mixed layer 3 has the same composition ratio of the metal having free electrons and the inorganic or organic substance throughout the entire layer. This mixed layer 3 functions as a metal thin film having substantially the same thickness as a metal thin film having a certain thickness, and at the same time functions as a protective layer or an intermediate layer having a certain thickness. Here, the thickness of the metal thin film having substantially the same function as that of the mixed layer 3 is α2
When the thickness of a protective layer or an intermediate layer having substantially the same function as that of the mixed layer 3 is β2, α2 and β2
Are thicker than the mixed layer 3 shown in the figure. That is, according to the present invention, the same function as a metal thin film having a certain thickness and a protective layer or an intermediate layer having a certain thickness is realized by the mixed layer 3 which is thinner than the sum of the thicknesses. As a result, as in the case of the first aspect according to the present invention, the same effect as when the test substance is substantially brought closer to the metal thin film can be obtained, and the advantage that the measurement sensitivity can be increased is enjoyed. be able to.

In the second embodiment of the present invention, the form of mixing the metal having free electrons and the inorganic or organic substance in the mixed layer is not particularly limited as long as the effects of the present invention can be obtained. For example, as a form of mixing, a form in which metal atoms and inorganic or organic molecules are mixed at an atomic and molecular level, a form in which metal particles and inorganic or organic particles are homogeneously mixed, and the like are all possible. Included in the present invention. In the latter case, the form is schematically shown in FIG. FIG. 6 schematically shows a cross section near the upper surface 21 of the mixed layer 3 shown in FIG. In the mixed layer 3 shown in the figure, metal particles 61 having free electrons and inorganic or organic particles 62 are mixed. In this embodiment, the upper surface 21 has a configuration in which metal particles 61 and inorganic or organic particles 62 are two-dimensionally arranged with a certain mixing ratio.

According to the third aspect of the present invention, as shown in FIG. 7, a metal having free electrons and an inorganic or organic substance are each formed of a thin film laminate or a thin film parallel to a depth direction of a mixed layer. A mixed layer in the form of an aggregate of columns is provided. FIG. 7 (a) schematically shows a cross section near the upper surface 21 of the mixed layer according to the third embodiment of the present invention. The mixed layer 3 shown in FIG. 7A is formed by assembling a metal phase 71 having free electrons and an inorganic or organic phase 72 in parallel in the depth direction of the mixed layer. When each of the phases 71 and 72 is a thin film, the mixed layer 3 viewed from the upper surface 21 has a structure shown in FIG. That is, the mixed layer 3 has a configuration in which the thin films 71 and 72 are stacked. When the phases 71 and 72 are columnar, the mixed layer 3 viewed from the upper surface 21 has the structure shown in FIG. That is, the mixed layer 3
Has a structure in which a metal column 71 having free electrons and an inorganic or organic column 72 are assembled. Even when such a thin film is in a laminated or columnar form, the mixed layer functions not only as a metal thin film but also as a protective layer or an intermediate layer. Therefore, in the present invention, the term “comprising both” refers only to an embodiment in which two components are physically mixed and contained in the same layer as in the first and second embodiments of the present invention. However, as in the case of the third embodiment of the present invention, each of the two components also means a laminated body of thin films or an aggregate of columnar bodies.

In this embodiment, the thickness of the mixed layer 3 may be appropriately determined in consideration of various conditions, but the lower limit is preferably about 10 nm, more preferably about 20 nm, and most preferably about 30 nm. It is. The upper limit is preferably about 100 nm, more preferably about 80 nm, and most preferably about 70 nm.

According to still another aspect of the present invention, a metal layer having free electrons may be provided between the mixed layer and the support. The sensor element of this embodiment is shown in FIGS. In the embodiments shown in these figures, a metal layer 81 or 91 having free electrons is provided on a support 2.
Then, the mixed layer 3 is provided thereon. In the embodiment of FIG. 8, the mixed layer 3 is the mixed layer according to the first embodiment of the present invention. That is, the metal has a gradient composition in which the metal content decreases in proportion to the distance from the support 2. In the embodiment shown in FIG. 9, the mixed layer 3 is the mixed layer according to the second embodiment of the present invention. That is,
In the mixed layer 3, the composition ratio of the metal having free electrons and the inorganic or organic substance is the same throughout the entire layer.

The metal layer 81 or 91 in the embodiment shown in FIGS. 8 and 9 functions as a metal thin film, and at the same time, the mixed layer 3 thereon also functions as a metal thin film. Therefore, the metal layers 81 and 91 play a part of the function of the metal thin film performed by the mixed layer 3. As a result, the mixed layer 3 can be made thinner. Further, since the mixed layer 3 can be made thinner, the test substance can be brought closer to the metal thin film, so that the advantage that the measurement sensitivity can be further increased can be enjoyed.

In this embodiment, the metal layer 81 or 91
The thickness of may be appropriately determined in consideration of various conditions, particularly the metal species used for exciting surface plasmon resonance,
The lower limit is preferably about 10 nm, more preferably 2 nm.
It is about 0 nm, most preferably about 30 nm. The upper limit is preferably about 100 nm, more preferably about 80 nm, and most preferably about 70 nm.
nm.

In the embodiment shown in FIG. 8, the thickness of the mixed layer 3 may be appropriately determined in consideration of various conditions, but the lower limit is preferably about 0.2 nm, more preferably about 0.5 nm. And most preferably about 1 nm. The upper limit is preferably about 100 nm, more preferably about 70 nm, and most preferably 60 nm.
nm.

Further, in the embodiment shown in FIG. 9, the thickness of the mixed layer 3 may be appropriately determined in consideration of various conditions.
The lower limit is preferably about 0.1 nm, more preferably about 0.3 nm, and most preferably about 0.5 nm. The upper limit is preferably about 100 nm, more preferably about 70 nm, and most preferably about 60 nm.

In the present invention, the metal constituting the mixed layer 3 is not particularly limited as long as it has a so-called free electron, but is preferably selected from the group consisting of gold, silver, copper, aluminum and platinum. Are included.

As described later, the inorganic substance or the organic substance is used for its purpose, that is, whether it is a component for protecting the metal, a component for supporting the sensitive layer, or a support for supporting the sensitive layer. It may be appropriately determined according to whether or not the component carries the auxiliary layer. On the other hand, in the present invention, since the inorganic substance or the organic substance must coexist with the metal in the mixed layer, the relationship with the metal must be considered. Generally preferred components of this inorganic material include a material selected from the group consisting of silicon, titanium, aluminum, and tantalum, its oxide, nitride, or fluoride;
Or magnesium fluoride. According to the most preferred embodiment of the present invention, the metal contained in the mixed layer is gold or silver, and the inorganic substance contained in the mixed layer is silicon oxide or titanium oxide.

The support is not particularly limited as long as it has an adhesive property to the mixed layer or the metal layer and has a light transmitting property. However, the refractive index of this support is
It must be greater than the refractive index of the medium of the test substance in contact with the side. The support is preferably made of glass, plastic, various glasses, plastics such as acrylic, polycarbonate, polystyrene, norbornene, and the like, ionic crystals, molecular crystals, and the like. Further, the thickness of the support may be appropriately determined,
The lower limit is preferably about 100 μm, more preferably about 200 μm, and most preferably about 300 μm.
m. The upper limit is preferably about 2 mm, more preferably about 1 mm, and most preferably about 0.8 mm.

Further, the support is a prism 5, a diffraction grating,
It may be formed integrally with an optical fiber or the like.

Usage of Sensor Element The sensor element according to the present invention is used as follows.

First, the first mode is one in which the sensor element 1 is used without providing another layer on the upper surface 21 of the mixed layer. In this embodiment, the mixed layer has both functions of a metal thin film and a protective layer for protecting the metal thin film. Specifically, the test substance is brought into contact with the surface 9 of the mixed layer in a manner as shown in FIG. Here, the test substance includes a medium and a medium, and the medium may be any of a liquid, a gas, and a gel. Examples of typical test substances are solutions, gas mixtures. Depending on the refractive index of the test substance in contact with the surface 9, the intensity at certain angles of incidence decreases. Further, the refractive index of the test substance changes depending on the physical properties or composition of the test substance, for example, the concentration of the solute. Therefore, a calibration curve indicating the relationship between the concentration of a predetermined solute and the incident angle at which the intensity decreases is created in advance, and the refractive index of a substance in contact with the surface 9, that is, the concentration of a certain solute can be known based on the calibration curve. Becomes

In this embodiment, since the mixed layer also serves as a protective layer, an inorganic or organic substance is a component for protecting a metal. Specific examples thereof include simple substances such as silicon, titanium, aluminum, and tantalum; oxides, nitrides, and fluorides thereof; magnesium fluoride, Teflon, polycarbonate, PVC, polystyrene, organic polymers such as norbornene, and cellulose derivatives. And polysaccharides represented by

The second mode of use of the sensor element according to the present invention is that a sensitive layer comprising a capture substance having an affinity for a target substance is further carried on the mixed layer. The structure of the sensor element used in this embodiment is schematically shown in FIG. In the figure, a sensitive layer 101 is provided on an intermediate layer 3. This sensitive layer is made of an inorganic or organic substance existing on the surface 9 of the mixed layer 3.
It is formed by carrying a capture substance physically or chemically. A sample containing a target substance is brought into contact with the surface 102 of the sensitive layer 101. When the target substance is present in the sample, it is captured by a substance having an affinity for the target substance in the sensitive layer. The degree of this capture should be proportional to the affinity between the two and the concentration of the target substance in the sample. Therefore, the amount of the target substance captured in the sensitive layer 101 depends on the concentration of the target substance in the sample. When the target substance is captured by the sensitive layer 101, the target substance is present in the vicinity of the surface 9 of the mixed layer 3;
Depending on its abundance, the intensity at certain angles of incidence decreases.
Therefore, for the target substance, a calibration curve indicating the relationship between the predetermined concentration and the incident angle at which the intensity decreases is created in advance, and the concentration of the target substance in the sample that comes into contact with the surface 9 can be known based on the calibration curve. Become.

Here, specific examples of the capture substance having an affinity for the target substance include proteins, peptides, antibiotics, dyes, nucleic acids, pesticides, microorganisms, hormones, or viruses, or antigens of their components; Polyclonal, monoclonal, or recombinant antibodies that recognize an antigen; or biologically-related substances such as enzymes, lectins, nucleic acids, or receptor ligands involved in signal transmission in vivo, which destroy the active site and function only as a binding site. Can be By using such a bio-related substance, the sensor element according to the present invention can measure, for example, albumin and hemoglobin if it is a protein, and hCG and LH if it is a hormone, for a clinical sample. It becomes possible. Also, when targeting environmental samples, biosensors that can detect pathogenic microorganisms represented by Salmonella, pathogenic E. coli, etc. by detecting water, pesticide residues in foods, antibiotics, etc., and recognizing surface antigens Available as

The trapping substance is carried on an inorganic or organic substance in the mixed layer, and it may be either physically carried or chemically carried. That is, the trapping substance may be physically adsorbed to an inorganic or organic substance in the mixed layer, or may be chemically bonded via a chemical bond. In the case of a chemical bond, the functional group of the inorganic or organic substance may be bonded to the functional group of the capturing substance using the functional group, or may be bonded to the inorganic or organic substance via, for example, a silane coupling agent. .

In this embodiment, specific examples of the inorganic or organic substance in the mixed layer include simple substances such as silicon, titanium, tantalum, and aluminum, oxides, nitrides, fluorides, or magnesium fluorides thereof, and further, Teflon, Examples thereof include organic polymers such as polycarbonate, PVC, polystyrene, and norbornene, and polysaccharides represented by cellulose derivatives. In particular, the use of silicon oxide makes it easy to form a mixed substance with a metal and has a low refractive index (the refractive index of silicon dioxide is 1.5 to 1.6).
Is much lower than the refractive index of titanium dioxide (2.6-2.8), the material is stable, and it can be easily activated by a known method such as silanization to form a sensitive layer.

Further, according to a preferred embodiment of the present invention, an additional layer is provided between the sensitive layer and the mixed layer in the second mode of use, so that the sensitive layer of the mixed layer can be supported more reliably and stably. You may. Specifically, a material capable of physically or chemically supporting the supporting auxiliary layer is selected as an inorganic or organic material constituting the mixed layer, and the mixed layer is formed.
At the same time, the supporting auxiliary layer is formed of a material capable of physically or chemically supporting a capturing substance having an affinity for the target substance. As a result, on the mixed layer, via the supporting auxiliary layer,
It becomes possible to carry a sensitive layer comprising a capture substance having an affinity for the target substance.

The substance constituting such a supporting auxiliary layer includes a polysaccharide or an organic polymer containing a group selected from the group consisting of hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, and vinyl groups, or Copolymers consisting of monomers comprising the groups are mentioned. Further examples of these include:
Carboxymethylated dextran, hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, dextran containing any of vinyl groups by derivatization, agarose, carrageenan, alginic acid, starch, polygalacturonic acid, polysaccharides such as cellulose, Examples thereof include organic polymers such as polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polyethylene glycol, and polyacrylamide, and copolymers of monomers constituting these polymers. Preferred specific examples of inorganic or organic substances capable of supporting such a supporting auxiliary layer include simple substances such as silicon, titanium, tantalum, and aluminum, and oxides, nitrides, fluorides, and magnesium fluorides thereof. Inorganic substances, plastics such as Teflon, polycarbonate, PVC, polystyrene, norbornene and the like, and organic substances such as silane coupling agents.

The thickness of the supporting auxiliary layer is preferably such that its existence does not affect the sensitivity of the sensor.
The thickness is preferably about 0 nm, more preferably 10 nm.
About 100 nm.

The supporting substance of the supporting auxiliary layer may be supported not only on the surface thereof but also inside the supporting auxiliary layer, and this embodiment is considered to be preferable.
When the target substance can diffuse in the supporting auxiliary layer and reach near the mixed layer surface, a reaction between the target substance and the capture substance can be caused in a place closer to the mixed layer,
This is because measurement with higher sensitivity becomes possible.

In any of the second and third modes of use, the capture substance having an affinity for the target substance can be selected by the user and carried on the mixed layer prior to use. That is, the sensor element is supplied to the user in a state in which the capture substance is not supported, and the user can select the capture substance as appropriate and use the capture element.

The sensor element according to the present invention is used in the manner described above. From another viewpoint, according to the present invention, a measuring method and a sensor device using the sensor element are provided.

First, corresponding to the first mode of use, according to the present invention, there is provided a sensor device capable of measuring a change in the physical property or composition of a test sample. This device irradiates the sensor element according to the present invention, the means for bringing the mixed layer of the sensor element into contact with the sample, and the interface between the support of the sensor element and the mixed layer or the metal layer through the support to irradiate light. Possible light irradiation means and means for measuring a reflection angle of light whose intensity has changed in reflected light reflected at the interface between the support of the sensor element and the mixed layer or the metal layer are provided.

According to the present invention, corresponding to the first mode of use, a method for measuring a change in the physical properties or composition of a test sample is provided. This method comprises the steps of: contacting the sample with the mixed layer of the sensor element according to the present invention; and irradiating light through the support at the interface between the support and the mixed layer or the metal layer of the sensor element, Measuring the angle of reflection of the light reflected by the interface between the support of the sensor element and the mixed layer or the metal layer, the intensity of which has changed.

Further, corresponding to the second mode of use,
According to the present invention, a sensor device capable of quantifying a target substance in a sample is provided. This device comprises a sensor element having a sensitive layer formed on a mixed layer of the sensor element according to the present invention, optionally via a supporting auxiliary layer, a means for bringing the sensitive layer of the sensor element into contact with a sample, and a support for the sensor element. And at the interface between the mixed layer and the metal layer, the light irradiating means capable of irradiating light by passing through the support, and the reflected light reflected at the interface between the support of the sensor element and the mixed layer or the metal layer, Means for measuring a reflection angle of light having a change in intensity.

According to the present invention, there is provided a method for quantifying a target substance in a sample according to the second aspect of the present invention, wherein a supporting auxiliary layer is optionally provided on the mixed layer of the sensor element according to the present invention. Contacting the sample with the sensitive layer of the sensor element having a sensitive layer formed therethrough, and irradiating light to the interface between the support and the mixed layer or the metal layer of the sensor element through the support. Measuring the angle of reflection of the light reflected at the interface between the support of the sensor element and the mixed layer or the metal layer, the intensity of which has changed.

In the second mode of use according to the present invention, the target substance remains on the sensor element surface after being captured by the capture substance. The captured target substance can be eliminated under appropriate conditions. For example, when an antigen-antibody reaction is used, the antigen and the antibody can be separated from each other by washing with a buffer having a certain pH range or with an acid. The sensor element from which the target substance has been removed from the surface by such a method can be used again for measurement.

Manufacture of the sensor element The sensor element according to the invention is preferably manufactured as follows.

First, a support is prepared. The surface of the support is preferably subjected to a washing operation before production. The washing method may be appropriately selected depending on the material of the support. In the case of glass, an acid, a detergent, a planisolution may be used, and ultrasonic cleaning may be used together.

A mixed layer is formed on a support whose surface has been cleaned. The formation of the mixed layer can be preferably carried out by a method in which a metal layer and a layer made of an inorganic substance or an organic substance are individually formed, and then a method of mixing the two or a method of co-evaporating a metal and an inorganic substance or an organic substance. .

In the former case, when an inorganic substance is used, thermal annealing is preferable, and when an organic substance is used, plasma treatment is preferable. Alternatively, the mixed layer can be formed by applying a voltage, a pressure, or the like. More specifically, it is as follows.

In the case of thermal annealing, first, a metal having free electrons is laminated on a support, and an inorganic or organic substance is laminated thereon. When heating the obtained laminate,
The metal layer and the inorganic or organic layer penetrate into each other, and eventually become a mixed layer containing both the metal and the inorganic or organic substance. The thermal annealing condition may be appropriately determined in consideration of the type and thickness of the metal and the type and thickness of the inorganic or organic substance.
It is preferable to heat at 00 ° C to 800 ° C for 1 minute to 24 hours, more preferably at 150 ° C to 500 ° C for 3 minutes to 2 hours.
Heat for hours.

In the case of plasma treatment, a laminate is first prepared in the same manner as in the case of thermal annealing, and corona discharge or glow discharge is performed on the laminate. As a result, a region including the uppermost portion of the metal layer is brought into a plasma state, the material is rearranged, and a mixed layer containing both a metal and an inorganic or organic material is obtained. In this case, the conditions such as the degree of vacuum, the type of atmospheric gas, the temperature discharge intensity, and the time may be appropriately determined in consideration of the type and thickness of the metal, and the type and thickness of the inorganic or organic substance. Is preferable.

The co-evaporation can be performed by using, for example, multi-target sputtering or multi-target CVD. That is, the composition of the mixed layer is controlled by independently controlling the deposition speed of each component.

When the metal and the inorganic or organic substance have a uniform composition, the metal and the inorganic or organic substance can be mixed in advance, and the mixture can be used as a target for vapor deposition.

When a metal layer is formed between the support and the mixed layer, the metal layer can be formed by a known thin film forming method, for example, vacuum evaporation or sputtering evaporation. Further, when the adhesiveness between the metal and the support is poor, it is preferable to provide an adhesive layer before vapor deposition according to a conventional method.

[0062]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Formation of Gradient Mixed Substance by Annealing A cover glass (size 2 × 4 cm) as a support was prepared and ultrasonically cleaned in the order of dilute nitric acid, a neutral detergent, and ultrapure water. A 2 nm thick Cr adhesive layer was applied on this support by magnetron sputtering,
A gold thin film having a thickness of nm was deposited. Subsequently, a silicon film having a thickness of 2 nm was formed by the same magnetron sputtering method. It was stored in an argon atmosphere until annealing to prevent silicon oxidation.

Next, the substrate obtained above was placed in an electric furnace,
Annealing at 200 ° C for 10 minutes in a nitrogen atmosphere,
A mixed substance having a gradient structure was formed. Thereafter, the device is stored in an oxygen-containing atmosphere for one day to oxidize the surface silicon to silica.

Example 2 Formation of a gradient mixed substance by co-evaporation A cover glass washed in the same manner as in Example 1 (size 2 × 4
cm) as a support, and ultrasonic cleaning was performed in the order of diluted nitric acid, a neutral detergent, and ultrapure water. On this support, two targets, silver and silicon dioxide, were mounted in a multi-target sputtering apparatus, and a silver thin film having a thickness of 45 nm was first deposited with a silver target. Subsequently, the sample side was rotated, and silver and silicon dioxide were co-sputtered at predetermined rates. During the progress of sputtering, the sputtering speed of the silver target was gradually decreased while the sputtering speed of the silicon dioxide target was gradually increased. As a result, a mixed layer with a thickness of 5 to 10 nm having a tilted structure in which silver was hardly exposed on the surface could be formed.

Example 3 Production of Immunosensor for Measurement of Albumin (Part 1) The substrate prepared in Example 1 or Example 2 was treated with 0.5 (v /
v) 5% glycidoxypropyltrimethoxy-silane solution (solvent isopropyl alcohol) at room temperature
The surface was silanized for minutes. Then, it was washed with water. The silanized substrate was shaken in a 23% (w / v)% dextran (molecular weight 500,000) solution for 20 hours to form a dextran gel layer, and then washed with a large amount of water. The immobilized dextran layer was shaken in 1M iodoacetic acid for 48 hours, dextran was carboxymethylated (CM), and then washed with a large amount of water.

The CM-dextran gel was added to 0.1 M N-
After activation with hydroxysuccinimide and dimethylaminopropylcarbodiimide (EDC), the albumin antibody was immobilized.

The obtained antibody-immobilized glass plate was 7 × 7 mm
To obtain a sensor element.

Example 4 Production of Immunosensor for Measurement of Albumin (Part 2) A dextran gel layer was formed by a method different from that of Example 3. First, 1M bromoacetic acid and 2N NaOH
Was prepared. To 30 ml of this solution, 10 g of dextran (molecular weight 500,000) was added and dissolved, followed by stirring at room temperature for 20 hours to convert the dextran into CM. Thereafter, the solution was neutralized, purified by dialysis with a large amount of water, freeze-dried, and stored until immediately before use.

The substrate prepared in Example 1 or 2 was placed in a 0.5% (v / v)% glycidoxypropyltrimethoxy-silane solution (solvent: isopropyl alcohol), and the surface was treated at room temperature for 5 minutes. After silanization, it was washed with water.

The silanized substrate was shaken for 20 hours in a dextran CM solution prepared above at 23 (w / v)% to form a CM dextran gel layer on the substrate. Then, it was washed with a large amount of water.

Example 5 Human Serum Albumin (HSA)
Detection of human serum albumin (HS
A) The sensor element on which the antibody was immobilized was mounted on the device shown in FIG. 1 and HSA was measured.

First, an appropriate buffer is passed through the detection surface.
The resonance angle at that time was measured and set as a baseline.

Next, the HSA solution was brought into contact with the sensor element surface for a certain period of time to bind the HSA antibody and HSA on the element surface. Then, the excess HSA was washed out using an appropriate buffer solution, and the resonance angle was measured. The difference between this resonance angle and the resonance angle at the baseline is the amount of H bound to the antibody on the sensor element surface.
The resonance angle changes due to the SA amount. H bound to the antibody
SA could be dissociated with a suitable eluate (dilute hydrochloric acid or glycine buffer below pH 3.0), allowing reuse of the detection surface.

The change in the resonance angle in the above operation is shown in FIG.
1 as shown.

Further, the HSA concentration and the resonance angle were measured using several samples having known HSA concentrations. The result was as shown in FIG. By using this as a calibration curve, HSA measurement with unknown concentration can be performed.

Example 6 Formation of Homogeneous Mixed Substance by Co-Evaporation A cover glass washed in the same manner as in Example 1 (size 2 × 4
cm) as a support, and ultrasonically wash in the order of dilute nitric acid, a neutral detergent, and ultrapure water. On this support, two targets of gold and silicon dioxide are mounted on a multi-target sputtering apparatus, and a gold thin film having a thickness of 45 nm is first deposited by a gold target. Subsequently, the sputtering rate of each target of gold and silicon dioxide is adjusted to form a mixed layer having a ratio of gold: silicon dioxide of 7: 3 to 5 nm. As shown in FIG. 5 (b), the mixed layer obtained in this way is a mixture of gold particles and silica particles, and the surface thereof is composed of gold particles and silica particles of 7: 3.
Are arranged two-dimensionally with a mixing ratio of
Here, it is considered that the size of the gold particles and the silica particles obtained by sputtering is on the order of nanometers.

The substrate obtained as described above can be used as an immunosensor for albumin measurement according to Example 3 or 4.

REFERENCE EXAMPLE Influence of Metal Monolayer Thickness on Surface Plasmon Resonance
The sensor element was manufactured by changing to 5, 50, 55, 60, 65, and 70 nm.

These sensor elements were mounted on the apparatus shown in FIG. 1, water was prepared as a test sample, and the ratio of the reflection intensity at the resonance angle to the total reflection intensity was determined. Then, a relationship between the thickness of the metal layer of the sensor element and the ratio of the reflection intensity at the resonance angle, that is, a resonance curve was prepared. The result was as shown in FIG. As a result, 30-7
It was revealed that the reflectance was about 0.5 or less in the case of the metal monolayer of 0 nm.

[Brief description of the drawings]

FIG. 1 is a diagram of a sensor device using a surface plasmon resonance method using a sensor element according to the present invention.

FIG. 2 is a view showing a preferred example of a sensor element 1 according to the present invention. In the embodiment of this figure, the sensor element 1 is composed of a support 2 and a mixed layer 3 formed thereon.
Further, the mixed layer 3 has a gradient composition in which the metal content decreases in proportion to the distance from the support 2.

FIG. 3 is a view showing a sensor element in which a metal layer 31 and a protective layer or an intermediate layer 32 are independently provided on a support 2;

FIG. 4 shows another preferred example of a sensor element according to the present invention. In the embodiment of this figure, the mixed layer 3 is composed of a plurality of layers 41 to 47, and the metal content is constant in each of these layers, but the metal content is different between adjacent layers. , The support 2 as a whole
It is configured such that the content of metal decreases in proportion to the distance from the object.

FIG. 5 is a diagram showing another preferred example of the sensor element according to the present invention. This figure shows a mixed layer 3 in which the composition ratio of a metal and an inorganic or organic substance is the same throughout the entire layer.

6 is a diagram schematically showing a cross section near the upper surface 21 of the mixed layer 3 shown in FIG. The mixed layer 3 includes metal particles 5
1 and inorganic or organic particles 52 are homogeneously mixed.

FIG. 7 is a diagram showing another preferred example of the sensor element according to the present invention. FIG. 7A schematically shows a cross section near the upper surface 21 of the mixed layer according to this embodiment. The mixed layer 3 is composed of a metal phase 71 having free electrons and an inorganic or organic phase 72 gathered in parallel in the depth direction of the mixed layer. FIG. 7B is a diagram showing a case where each of the phases 71 and 72 is a thin film. FIG. 7C is a diagram showing a case where each of the phases 71 and 72 is a columnar body.

FIG. 8 is a diagram showing an embodiment in which a metal layer 81 is provided between a support 2 and a mixed layer 3 in the sensor element of FIG.

9 is a diagram showing an embodiment in which a metal layer 91 is provided between the support 2 and the mixed layer 3 in the sensor element of FIG.

FIG. 10 is a diagram showing one use mode of the sensor element according to the present invention. In the embodiment shown in the figure, a sensitive layer 101 is provided on the intermediate layer 3.

FIG. 11 is a diagram illustrating a change in resonance angle when an HSA solution, a cleaning solution, or an eluent is brought into contact with a sensor element according to the present invention, implemented in Example 5.

FIG. 12 shows some HS performed in Example 5.
It is a figure which shows the result of having measured the HSA density | concentration and the resonance angle using the sample whose A density | concentration is known. By using this as a calibration curve, HSA measurement with unknown concentration can be performed.

FIG. 13 is a diagram showing a resonance curve of a sensor element using surface plasmon resonance.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Jin Ohara 2-1-1 Nakajima, Kokurakita-ku, Kitakyushu-shi, Fukuoka Tochiki Kiki Co., Ltd. 2-1-1 Nakajima, Ward Totoki Co., Ltd.

Claims (28)

[Claims] 1. A light-transmissive support, comprising: a mixed layer formed on the support and containing both (a) a metal having free electrons and (b) an inorganic or organic substance. A sensor element utilizing surface plasmon resonance. 2. The sensor element according to claim 1, wherein said inorganic substance or organic substance is a component for protecting said metal. 3. The method according to claim 1, wherein the inorganic substance or the organic substance is capable of physically or chemically supporting a capture substance having an affinity for the target substance. As a result, the mixed layer has a capture substance having an affinity for the target substance. The sensor element according to claim 1, wherein the sensor element can support a sensitive layer containing a substance. 4. The method according to claim 1, wherein the inorganic or organic substance is capable of physically or chemically supporting the supporting auxiliary layer, and the supporting auxiliary layer physically or chemically supports a capturing substance having an affinity for a target substance. 2. The method according to claim 1, wherein the support layer is capable of supporting, and as a result, a sensitive layer including a capture substance having an affinity for a target substance can be supported on the mixed layer via a support auxiliary layer. The sensor element according to any one of the preceding claims. 5. The sensor according to claim 1, wherein the mixed layer has a gradient composition in which the content of the metal decreases in proportion to the distance from the support. element. 6. The sensor element according to claim 5, wherein the content of the metal in a portion of the mixed layer farthest from the support is 50% or less. 7. The sensor element according to claim 1, wherein the mixed layer has a substantially homogeneous composition. 8. The sensor element according to claim 1, wherein a metal layer having free electrons is provided between said support and said mixed layer. 9. The sensor element according to claim 8, wherein said mixed layer has a thickness of 100 nm or less. 10. The method according to claim 1, wherein said metal layer has a thickness of 10 nm or more and 100
The sensor element according to claim 9, which is equal to or less than nm. 11. The metal contained in the mixed layer is gold, silver,
The sensor element according to any one of claims 1 to 10, wherein the sensor element is selected from the group consisting of copper, aluminum, and platinum. 12. The inorganic material contained in the mixed layer is silicon,
2. A material selected from the group consisting of titanium, aluminum, and tantalum, or an oxide, nitride, or fluoride thereof, or magnesium fluoride.
The sensor element according to any one of claims 11 to 11. 13. The method according to claim 1, wherein the metal contained in the mixed layer is gold or silver, and the inorganic substance contained in the mixed layer is silicon oxide or titanium oxide. Sensor element. 14. The method according to claim 1, wherein a refractive index of said support is larger than a refractive index of a medium containing said target substance.
14. The sensor element according to claim 13. 15. A change in physical properties or composition of a test sample in contact with the mixed layer appears as a change in optical properties of the surface of the mixed layer or the metal layer in contact with the support.
The sensor element according to claim 1, 2, 5 to 14. 16. The method according to claim 1, wherein the presence or absence of a reaction between the target substance and the capturing substance carried on the mixed layer is represented as a change in the optical properties of the surface of the mixed layer or the metal layer in contact with the support. Item 15. The sensor element according to any one of Items 1, 3 to 14. 17. A sensitive layer comprising a capture substance having an affinity for a target substance is further provided on the mixed layer of the sensor element according to any one of claims 1, 3 to 16, Sensor element. 18. A support auxiliary layer is provided on the mixed layer of the sensor element according to any one of claims 1, 3 to 15, and further has an affinity for a target substance on the support auxiliary layer. A sensor element, further comprising a sensitive layer containing a capturing substance. 19. The supporting auxiliary layer comprises a polysaccharide or an organic polymer comprising a group selected from the group consisting of hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy and vinyl groups, or said group. 19. The sensor element according to claim 18, which is a copolymer composed of a monomer. 20. The capturing substance may be a protein, a peptide, an antibiotic, a dye, a nucleic acid, a pesticide, a microorganism, a hormone, or a virus, or an antigen of a component thereof; a polyclonal, monoclonal, or recombinant that recognizes these antigens. The sensor element according to any one of claims 16 to 19, wherein the sensor element is an antibody; or an enzyme, a lectin, a nucleic acid, or a receptor ligand involved in signal transmission in a living body, which destroys an active site and has only a binding site function. . 21. The method for manufacturing a sensor element according to claim 1, wherein a metal having free electrons is laminated on a support, and a layer made of an inorganic substance or an organic substance is formed thereon. And then subjecting to thermal annealing to form a mixed layer containing both the metal and the inorganic or organic material. 22. The method for manufacturing a sensor element according to claim 1, wherein a metal having free electrons is laminated on a support, and a layer made of an inorganic substance or an organic substance is formed thereon. And then mixing both layers by a plasma treatment to form a mixed layer containing both the metal and the inorganic or organic substance.
Method. 23. The method for manufacturing a sensor element according to claim 1, wherein a metal having free electrons and an inorganic or organic substance are co-deposited on a support, and And a step of forming a mixed layer containing both the inorganic and organic substances. 24. A layer comprising a metal having free electrons is formed on a support prior to the step of forming a mixed layer on a support, and the mixed layer is formed on the metal layer.
The method according to any one of the preceding claims. 25. A sensor device capable of measuring a change in physical property or composition of a test sample, wherein: the sensor element according to claim 1; a mixed layer of the sensor element and the sample; A light irradiating means capable of irradiating the interface between the support of the sensor element and the mixed layer or the metal layer and transmitting light through the support; and a support and the mixed layer or metal of the sensor element. A means for measuring a reflection angle of light having a change in intensity in reflected light reflected at an interface with the layer. 26. A sensor device capable of quantifying a target substance in a sample, wherein the sensor element according to claim 16 is in contact with a sensitive layer of the sensor element and the sample. Means for causing, at the interface between the support of the sensor element and the mixed layer or the metal layer, light irradiating means capable of irradiating light by passing through the support, and the support of the sensor element and the mixed layer or the metal layer; A means for measuring the angle of reflection of the light whose intensity has changed in the light reflected at the interface. 27. A method for measuring a change in physical properties or composition of a test sample, comprising: contacting the mixed layer of the sensor element according to any one of claims 1 to 15 with the sample; A step of irradiating the interface between the support of the sensor element and the mixed layer or the metal layer with light passing through the support, and reflecting light reflected at the interface between the support of the sensor element and the mixed layer or the metal layer; Measuring the angle of reflection of the light that has undergone a change in intensity. 28. A method for quantifying a target substance in a sample, comprising: contacting the sample with the sensitive layer of the sensor element according to claim 16; At the interface between the support and the mixed layer or the metal layer, the step of irradiating light by passing through the support, and in the reflected light reflected at the interface between the support and the mixed layer or the metal layer of the sensor element, Measuring the angle of reflection of the light that has undergone the change in intensity.