JPH08193948A - Exciting structure for surface plasmon resonance phenomenon and biosensor - Google Patents

Abstract

PURPOSE: To enhance sensitivity of a sensor in the measurement by making a film formed on the surface of a metallic thin film thin and uniform. CONSTITUTION: When a thin film of organic compound is bonded onto the surface of a thin metallic film (Au thin film), an organic compound represented by a chemical structural formula of A-B-C is employed, where A represents disulfide of five-membred ring, B represents a butyl group of 4C, and C represents a hydroxy group. For example, the organic compound is caoutchouc acid where a carboxybutyl group is bonded to disulfide of five-membered ring an the third substitution position thereof and the caoutchouc acid is bonded to a metallic thin film. In other words, two sulfur atoms fo the disulfide of five-membered ring in the caoutchouc acid are bonded to a gold atom on the surface of Au thin film under presence of a mixture solvent of ethanol/water.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a structure for exciting a surface plasmon resonance phenomenon by a tunnel effect of light on a light reflecting surface for totally reflecting incident light, and more specifically, for optically detecting a substrate to be measured. The present invention relates to an excitation structure of surface plasmon resonance phenomenon used in a biosensor.

[0002]

2. Description of the Related Art Generally, in a biosensor, when measuring a substrate to be measured which is a blood protein component such as a specific protein or antigen in blood, or a urine component such as glucose or ascorbic acid in urine, it is necessary to measure against these substrates. A biological substance having a discriminating function and causing a biochemical reaction with the substrate is used.
Then, various physicochemical displacements associated with the progress of this biochemical reaction are detected by the physicochemical device, and the specification of the measurement target substrate, its concentration, etc. are detected. For example, one that detects the substrate concentration through the electrode reaction of an electrode active substance that is consumed or produced by a biochemical reaction, or one that detects the enthalpy change that occurs with the progress of the biochemical reaction with a thermistor to detect the substrate concentration. These biosensors have been put to practical use since early on.

On the other hand, in recent years, attention has been paid to changes in the dielectric constant with the progress of biochemical reactions, and biosensors have been proposed for measuring a substrate to be measured in a solution to be measured using an optical device. (Japanese Patent Application Laid-Open No. 1-138443, Japanese Patent Application Publication No. 4-501462). In this biosensor, as an optical system, a light-transmitting light-transmitting medium that reflects light under a geometrical total reflection condition is provided on a light-reflecting surface provided with a metal thin film. An optical system that forms an evanescent wave coupling is used. The measuring principle is as follows.

When the light transmitting medium of the optical system for forming the evanescent wave coupling, for example, the p-polarized light is applied to the light reflecting surface at various incident angles satisfying the condition of total reflection,
A unique phenomenon occurs when the incident angle has a certain value. That is, p
When the light-reflecting surface is irradiated with the polarized light, an evanescent wave having a wave number having the incident angle θ as a variable is generated on the prism-side film surface of the metal thin film. Since metal can be regarded as a solid plasma, a surface plasmon wave as a wave of quantum theoretical charge density is tunneled to the anti-prism side film surface of the metal thin film (hereinafter simply referred to as a film surface). Caused by. This surface plasmon wave is generated as a wave between the metal thin film and the medium in contact with the film surface as a boundary surface.

When the incident angle θ has a certain value, that is, when the incident angle θ is the resonance angle, the evanescent wave and the surface plasmon wave resonate with their wave numbers matched, and the surface plasmon resonance phenomenon occurs. It is excited by the tunnel effect, and the energy of light is used as the excitation energy of surface plasmon waves. At this time, in terms of energy, there is a relation that the energy of the light incident on the light reflecting surface is equal to the sum of the energy used to excite the surface plasmon wave and the energy of the reflected light from the reflecting surface. Therefore, the state of changes in the reflection angle and the energy (light amount) is measured by using, for example, a multi-channel light receiving device, and the presence or absence of the surface plasmon resonance phenomenon,
By extension, the incident angle when the phenomenon occurs can be obtained. It should be noted that the p-polarized light may be performed while being received by the light-receiving device, and is not limited to being performed before being irradiated on the light reflecting surface as described above.

On the other hand, there is a correlation between the incident angle when the surface plasmon resonance phenomenon occurs and the refractive index of the medium, and this refractive index can be defined by the dielectric constant of the medium from Maxwell's equation, and the biochemistry by biological substances. There is a correlation between the progress of the static reaction and the dielectric constant. Therefore, the angle of incidence at that time is determined from the angle of reflection when the amount of reflected light suddenly decreases, and the degree of progress of the biochemical reaction by the biological substance, that is, the substrate concentration is calculated from the above correlations.

By the way, in such a biosensor, it is necessary to cause a biochemical reaction between the substrate and the biological substance in the vicinity of the surface of the metal thin film, specifically, in the evanescent region (about 100 nm) which causes the tunnel effect. There is. Therefore, it is an essential issue for this type of biosensor to immobilize a biological substance that has a function of discriminating against a substrate to be measured and that undergoes a biochemical reaction with the substrate on the film surface of a metal thin film in the evanescent region. Is. Therefore, various techniques have been conventionally proposed.

For example, in Japanese Patent Laid-Open No. 63-75542, a metal oxide thin film is formed on the surface of a metal thin film, the thin film is treated with a silane coupling agent, and an arbitrary substituent is added to the film surface of the thin film. A technique for introducing and immobilizing a biological substance on this substituent has been proposed. In addition, JP-A-4-351946
Then, a biological substance is immobilized on an LB film formed by the so-called LB method, specifically, the biological substance is adsorbed and immobilized on an arachidic acid monomolecular film on the water surface. There has been proposed a technique for copying (-B film) onto the film surface of a metal thin film. Further, in Japanese Patent Application Laid-Open No. 4-501605, X which is an asymmetric or symmetrical disulfide, R which is a saturated hydrocarbon chain and Y which is an active group such as hydroxyl are represented as a compound structural formula X-R-Y. It has been proposed to chemically bond an organic thin film of an organic compound to the surface of a metal thin film to immobilize a layer in which a biological substance is immobilized on the active group Y, a so-called ligand layer.

[0009]

In these biosensors, the measurement principle is to detect the change in the dielectric constant within the evanescent region on the film surface of the metal thin film. Therefore, in order to improve the measurement sensitivity and performance, it is necessary to cause the change in the dielectric constant with the progress of the biochemical reaction to be as uniform as possible near the film surface of the metal thin film. Therefore, it is the most important issue to improve the measurement sensitivity and performance to make the film interposed on the film surface of the metal thin film uniform and as thin as possible in order to fix the biological substance. In this case, the film interposed on the film surface of the metal thin film is a metal oxide thin film, an L-B film, or an organic thin film made of an organic compound X-R-Y in the above technique. Since the ligand layer is a layer itself that fixes a biological substance and a biochemical reaction proceeds in the layer to bring about a change in the dielectric constant,
Unlike the various thin films described above, it is irrelevant to the uniformity of the film.

However, in the technique of forming a thin film of a metal oxide on the film surface of a metal thin film, since the metal oxide is formed into a thin film by a method such as sputtering, the film thickness can be made uniform or thin. Is difficult. Further, in the technology using the LB film, because of the convenience of adopting the LB method, there is a need for facilities for preparing various buffer solutions and biological material-containing solutions and the like, and a processing step therefor, and thus lack of mass productivity. There is a problem that costs increase due to investment / maintenance of facilities or increase in man-hours.

On the other hand, an organic thin film made of an organic compound of X-R-Y causes less problems in terms of mass productivity and cost, but has the following problems due to the structure of the compound.

On the film surface of the metal thin film, as shown schematically in FIG. 6, each organic compound (X-
It can be said that RY) is bonded to the metal atom on the film surface of the metal thin film by a single sulfur atom (S). In order to stabilize an organic thin film having a uniform thickness with such a bond, it is essential that the respective organic compounds (X-R-Y) are aligned in order. Then, this organic compound (X-R-
In order to secure the alignment of Y), it is necessary that the saturated hydrocarbon chain which is R in the structural formula has a relatively large number of carbon atoms, specifically, a saturated hydrocarbon chain having 12 to 30 carbon atoms. There is. It is experimentally known that the alignment of the saturated hydrocarbon chain can be obtained only with the saturated hydrocarbon chain having a relatively large number of carbon atoms, because the chain structure has a carbon bond angle. It is presumed that the intermolecular affinity between adjacent saturated hydrocarbon chains acts.

Therefore, when the bond between the sulfur atom (S) and the metal atom on the film surface of the metal thin film is broken, the organic compound itself separates from the metal thin film surface, and eventually the organic thin film itself becomes a uniform film. Cease to exist. Therefore, in the case of continuously measuring the substrate while continuously supplying the solution containing the measurement target substrate to the ligand layer, the bond may be broken by the supply of the solution. Lack. Further, due to the fact that the saturated hydrocarbon chain has a relatively large number of carbon atoms, there is a limit to thinning it.

The present invention has been made to solve the above problems, and improves the measurement sensitivity of a sensor by reducing the thickness of a film interposed on the film surface of a metal thin film with a uniform film thickness.
The purpose is to improve the durability of the film.

[0015]

In order to achieve the above object, the means adopted in the excitation structure for the surface plasmon resonance phenomenon according to claim 1 is a tunnel of light in a light reflecting surface for totally reflecting incident light. A structure that excites a surface plasmon resonance phenomenon by an effect, a light transmission medium that forms a transmission path of incident light and light that reflects, and a light transmission surface formed on the surface of the light transmission medium. And a first organic thin film formed on the film surface of the metal thin film,
A second organic thin film formed on the film surface of the first organic thin film, wherein the first organic thin film has a compound structural formula of A
-B-C is an organic compound, and A in the compound structural formula is a multi-membered sulfide or disulfide containing a sulfur atom, and B in the compound structural formula is a saturated hydrocarbon chain. C in the compound structural formula is at least one substituent selected from a hydroxyl group, a carboxyl group, an amino group, an aldehyde group, an epoxy group, an active group such as a vinyl group and a halogen atom, and the first organic thin film Is
It is fixed to the metal thin film by a chemical bond between the sulfur atom in the multi-membered ring sulfide or disulfide forming A of the compound structural formula and the metal atom in the metal thin film, and the second organic thin film is crosslinked. Dextran, cellulose, starch, agarose, polyacrylamide,
The gist of the invention is a hydrogel composed of at least one compound selected from polyvinyl alcohol, polyacrylic acid, polyethylene glycol and polyvinyl acetic acid and / or a derivative of the compound.

In the excitation structure of the surface plasmon resonance phenomenon according to claim 2, A in the structural formula of the compound is 5 to 7
It was either a sulfide or a disulfide of the member ring.

In the excitation structure of the surface plasmon resonance phenomenon according to claim 3, B of the compound structural formula has 2 carbon atoms.
To 8 saturated hydrocarbon chains.

The means adopted in the biosensor according to claim 4 is the biosensor using the excitation structure of the surface plasmon resonance phenomenon according to any one of claims 1 to 3, wherein the second organic thin film is used. The hydrogel forming the is provided with a biological substance that has a function of discriminating against the substrate to be measured and that causes a biochemical reaction with the substrate, and the light from the light source is transmitted through the light transmission medium to the light reflection surface. It is provided with a light irradiating means for converging and irradiating, and a light receiving means for receiving the reflected light reflected by the light reflecting surface and emitted from the light transmitting medium to the outside, and detecting the light amount of the reflected light. And

[0019]

In the excitation structure of the surface plasmon resonance phenomenon according to claim 1 having the above structure, the first organic thin film is formed on the film surface of the metal thin film formed so that the surface of the light transmitting medium serves as the light reflecting surface. The second organic thin film is formed and provided in this order. First
The organic thin film of is composed of an organic compound represented by A-B-C as a compound structural formula, and a chemical bond between a sulfur atom in a multi-membered sulfide or disulfide that is A of the compound structural formula and a metal atom in a metal thin film. Adheres to the metal thin film. That is, each of the organic compounds forming the first organic thin film is bonded to the metal thin film by the two sulfur atoms in the multi-membered sulfide or disulfide as schematically shown in FIG. Therefore, the number of binding sites per organic compound increases, and the binding of the organic compound to the metal thin film, and thus the binding of the first organic thin film, becomes strong. Further, since the bond itself becomes strong, the alignment of the saturated hydrocarbon chain, which is B of the compound structural formula, is not essential for stabilizing the first organic thin film, and there is a limitation that the number of carbon atoms is increased. Absent. Therefore, the first organic thin film is made uniform and thin. Therefore, the change in the dielectric constant due to the progress of the biochemical reaction can be uniformly caused in the evanescent region extremely near the film surface of the metal thin film.

Thus, the strong binding of the sulfur atom-containing multi-membered ring sulfide or disulfide to the metal thin film means that, for example, a sulfur-containing 5-membered or 6-membered sulfide or disulfide compound is It is reported to be strongly adsorbed on metals such as RGNuzzo et al. JACS, 1983, 105, 4
481-3). Then, a 5-membered or 6-membered sulfide or disulfide is added to the solvent in an amount of 0.0001.
By dissolving at a concentration of 0.01 mol / liter and immersing the metal in the solution for several minutes to several days, a monomolecular layer can be formed, and an ultrathin film having a thickness of nm order can be realized.

In the surface plasmon resonance excited structure according to the first aspect, since the first organic thin film fixes A of the compound structural formula to the metal thin film, C of the compound structural formula is used.
And the substituent is the second organic thin film side. Then, in this second organic thin film, the unreacted active group in its own hydrogel is covalently bonded to the substituent represented by C of the compound structural formula through a hydrolysis, an esterification reaction, an oxidation reaction, or the like. Is bonded and fixed to the film surface of the organic thin film. For example, when the substituent represented by C in the compound structural formula is a carboxyl group, the second organic thin film is a hydrogel having a hydroxyl group. Then, the second organic thin film is bonded and fixed to the film surface of the first organic thin film by the esterification reaction between the hydroxyl group in the hydrogel and the carboxyl group which is C of the compound structural formula, and the first organic thin film is bonded and fixed. Is formed on the film surface of the metal thin film. In this case, the second organic thin film becomes a ligand layer by immobilizing various biological substances on the hydrogel.

Here, the metal thin film to which the first organic thin film is fixed can form a light-reflecting surface by forming it on the surface of the light transmitting medium, and the multi-membered ring sulfide represented by the compound structural formula A can be used. Alternatively, a thin film made of a metal capable of binding to a sulfur atom in disulfide may be used. Therefore, in consideration of the bond strength with the sulfur atom, the gold thin film is most suitable, but it is needless to say that the copper or silver thin film may be used.

In the excited structure of the surface plasmon resonance phenomenon according to claim 2, since A of the compound structural formula is either a sulfide or a disulfide of a 5- to 7-membered ring,
It can be composed of these sulfides or disulfides which are general-purpose compounds. In this case, for example, if a thiobutylic acid having a carboxybutyl group bonded to the 3-position of a 5-membered disulfide is used, the carboxylic acid can be easily introduced into the film surface of the metal thin film. In this case, the organic compound is a 5-membered ring disulfide, which is A in the compound structural formula, to which a carboxylic acid composed of B and C in the compound structural formula is bonded, and the substitution position is the 3-position. Further, B in the compound structural formula is a butyl group of a saturated carbon-hydrogen chain, and the terminal substituent which is C in the compound structural formula is a carboxyl group. In this case, 5
A membered ring sulfide, a 6-membered ring or a 7-membered ring sulfide or a disulfide, or C in the compound structural formula may be an amino group, a hydroxyl group or the like, an aldehyde group, an epoxy group, a vinyl group or a halogen atom. Further, the substitution position of B in the compound structural formula is not limited to the 3-position, and the substitution position may be the 4-position or the like.

In the excitation structure of the surface plasmon resonance phenomenon of claim 3, since B of the compound structural formula is a saturated hydrocarbon chain having 2 to 8 carbon atoms, the organic compound defined by this carbon atom number Has a short chain length, which leads to thinning of the first organic thin film. In this case, when the number of carbon atoms is about 4, it is desirable because in addition to thinning the film, in addition to imparting appropriate mobility to the substituent of C in the compound structural formula. Further, it is preferable that B in the compound structural formula is a saturated hydrocarbon chain having 2 to 8 carbon atoms, because the raw materials thereof can be easily obtained and synthesized.

In the biosensor according to the fourth aspect, the second organic thin film serves as a ligand layer by immobilizing a biological substance on the hydrogel forming the second organic thin film. In this case, the immobilization of the biological substance on the hydrogel is performed by converting an unreacted hydroxyl group in the hydrogel into a carboxylic acid or an amino group and then binding the biological substance such as an enzyme or an antibody.

In this biosensor, the first organic thin film fixed on the film surface of the metal thin film of the excitation structure of the surface plasmon resonance phenomenon used is made uniform and thin,
The change in the dielectric constant due to the progress of the biochemical reaction occurs uniformly in the evanescent region in the very vicinity of the film surface of the metal thin film. Therefore, the surface plasmon resonance phenomenon in which the evanescent wave and the surface plasmon wave sandwiching the metal thin film resonate when their wave numbers match each other is easily excited.

In this case, in the biosensor having the above structure, the light from the light source is incident on the light transmitting medium, is totally reflected by the light reflecting surface, and is emitted from the light transmitting medium until received. The behavior of light during the period and the substrate measurement method are the same as the conventional one.

[0028]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, preferred embodiments of the surface plasmon resonance phenomenon excitation structure and a biosensor using the same according to the present invention will be described with reference to the drawings. First, the configuration of the biosensor 10 will be described.

As shown in FIG. 2, which is a schematic side view of the sensor, the biosensor 10 is provided with a prism 12 made of optical glass, and a sample plate 16 is provided on the upper surface of the prism 12 with a matching oil 14 interposed. It has been placed. The sample plate 16 is formed of the same optical glass as the prism 12, and its refractive index is the same as that of the prism 12. Also, matching oil 14
Has a refractive index of prism 12 or sample plate 16
It is an oil of the same degree as, and functions to match the refractive index between the prism 12 and the matching oil 14. Therefore, gel, hydrophobic polymer, or the like can be used instead of the matching oil 14 as long as it can fulfill the function.

A gold thin film (Au thin film) 18 having a film thickness of 50 nm is formed on the upper surface of the sample plate 16 by vapor deposition. The Au thin film 18 is formed on the upper surface of the sample plate 16.
The light reflection surface 20 is a total reflection surface that reflects light under the geometrical total reflection condition over the vapor deposition range. The sample plate 16 and the Au thin film 18 form evanescent wave coupling on the light reflecting surface 20. Further, on the film surface of the Au thin film 18, a ligand layer 22 having a function of discriminating against a substrate to be measured and immobilizing a biological substance which causes a biochemical reaction with the substrate is formed with an organic thin film layer 26 interposed. ing. The ligand layer 22 and the organic thin film layer 26 will be described later. The ligand layer 22 is
The front side and the back side of the paper are divided into two, the front side ligand layer 22 is a ligand layer on which an active biological substance is immobilized, and the other back side ligand layer 22 is a deactivated biological substance. It is a fixed ligand layer. That is, the front side ligand layer 22 is used as the substrate measurement sensor section, and the back side ligand layer 22 is used as the correction sensor section.
Is used. In this case, the biological material is inactivated by a strong acid or a strong alkali, or by electron beam irradiation such as ultraviolet rays, ultrasonic treatment, or heat treatment at about 70 ° C.

In addition, the biosensor 10 includes a light source (not shown) that linearly emits light of a single wavelength such as an LED (light emitting diode), and irradiates the prism 12 with light from this light source. A condenser lens 24 is arranged between the light source and the prism 12 in front of the prism 12, and a p polarizing plate (not shown) is arranged between the light source and the condenser lens 24. The position of the condenser lens 24 is adjusted so that the light passing through the condenser lens 24 is condensed in a line on the light reflection surface 20. Therefore, the light emitted from the light source reaches the condenser lens 24 after being p-polarized, and thereafter passes through the prism 12 as p-polarized light and is reflected by the light reflecting surface 20.
The light is focused in a line. Therefore, on the light reflecting surface 20, the light that is p-polarized by the p-polarizing plate and is a wave in the incident surface is the focal length F and the aperture length D of the condenser lens 24 and the angle of the optical axis of the condenser lens 24. It arrives at an incident angle (θ1 to θ2) within a predetermined range determined by the above.

A CCD image pickup device 28 for detecting the amount of received light and converting it into an electric signal is arranged on the side where the reflected light totally reflected by the light reflecting surface 20 is emitted from the prism 12. The CCD image pickup device 28 is a linear image sensor in which 2048-bit light receiving elements are arranged in a line. Therefore, the light (p-polarized light) totally reflected by the light reflecting surface 20 passes through the prism 12 and is received by the CCD image pickup device 28, and the CCD image pickup device 28 detects the amount of light. The reflected light reflected by the light reflecting surface 20 is a wave having an amplitude within the incident surface, similar to the incident light on the light reflecting surface 20, and in the CCD image pickup device 28, the amount of light for each reflecting angle, that is, the incident angle within the above range ( The amount of light for each θ1 to θ2) is the CCD
It is detected by the light receiving element in the light receiving range of the image pickup element 28.

In this case, on the film surface of the Au thin film 18 in the sample plate 16, the dielectric constant of the solution to be measured changes as described below depending on the presence or absence of the activity of the biological substance in the ligand layer 22 and stabilizes at the changed value. Then, this state is detected from each CCD image pickup device 28.

On the side of the ligand layer 22 to which the active biological substance is fixed, the biochemical reaction between the biological substance and the substrate to be measured proceeds to the extent defined by the substrate concentration, so that the dielectric constant of the solution to be measured is increased. The index, and thus its refractive index, changes as the biochemical reaction progresses and stabilizes at a value defined by the substrate concentration. At this time, the change in the dielectric constant of the solution to be measured, and thus in the refractive index, is different from that of the sample plate 16 and Au.
By the evanescent wave coupling formed with the thin film 18,
It is observed as a phenomenon of energy of reflected light when the surface plasmon resonance phenomenon occurs.

That is, the p-polarized light emitted from the light source is
The light is condensed by the condenser lens 24 and is in the above range (θ1 ~
At the incident angle of θ 2), the light reaches the light reflecting surface 20 where the evanescent wave coupling is formed. At this time, the p-polarized light incident at an incident angle of a certain angle θS1 within the above range is an evanescent wave on the light reflection surface 20 side film surface of the Au thin film 18 and a surface plasmon wave on the measured solution side of the Au thin film 18. And cause the surface plasmon resonance phenomenon by causing their wave numbers to resonate. When this surface plasmon resonance phenomenon occurs, the energy of the light whose incident angle is the resonance angle θS1 is used as the excitation energy of the surface plasmon wave, and the energy of the reflected light whose reflection angle from the light reflecting surface 20 is the resonance angle θS1 decreases. To do.

Therefore, the state of the amount of light received by the CCD image pickup device 28 which receives the reflected light from the light reflecting surface for each incident angle (θ1 to θ2) at the reflection angle of θ1 to θ2 is shown in FIG.
As schematically shown in FIG. 3, the energy (light amount) of the reflected light at the resonance angle θS1 is the lowest (FIG. 3 (A)).

On the other hand, since the biochemical reaction with the substrate to be measured does not proceed on the side of the ligand layer 22 to which the inactivated biomaterial is fixed, the dielectric constant of the solution to be measured, and hence its refractive index, is initially low. The value of is constant. However, the surface plasmon resonance phenomenon occurs due to the p-polarized light incident at an incident angle of a certain angle θS0, and the amount of received light in the CCD image pickup device 28 in this case is such that the energy of the reflected light at the resonance angle θS0 becomes the minimum (Fig. 3 (B)).

The incident angle on the horizontal axis in the schematic view of FIG. 3 corresponds to the arrangement of the 2048-bit light receiving elements in the CCD image pickup device 28. Then, the detection result of each light receiving element of the CCD image pickup element 28 forms the above light amount distribution.

Next, the ligand layer 22 and the organic thin film layer 2
The manufacturing process of No. 6 will be described. First, in the first step, the sample plate 16 is prepared in which the Au thin film 18 is vapor-deposited and formed in the above-described thickness over a predetermined range. The second that follows
In the step of, a thioctic acid having a carboxybutyl group bonded to the 3-position substitution position of the 5-membered disulfide is prepared, and the concentration of this thioctic acid is 2 mmol / in an ethanol / water mixed solvent (volume ratio 4: 1). Dissolve to liter to prepare a thiotic acid solution. After that, in the third step, the sample plate 16 was immersed in this prepared thiotic acid solution for 5 minutes, thoroughly washed with the above ethanol / water mixed solvent, and then washed with purified water. When the sample plate 16 is dipped, the Au thin film 18 may be dipped in a thiotic acid solution.

In the process of this third step, two sulfur atoms in the 5-membered disulfide of thiotic acid combine with the gold atoms on the Au thin film 18 film surface in the presence of the ethanol / water mixed solvent. In this case, the hydroxyl group (acidic hydroxyl group), which is the substituent at the terminal of the thiotic acid, does not bond with the gold atom on the surface of the Au thin film 18 due to its nature. Therefore, through the first to third steps, the 5-membered ring disulfide is designated as A in the compound structural formula, the butyl group having 4 carbon atoms is designated as B in the compound structural formula, and the hydroxyl group is designated as C in the compound structural formula. Then, the organic compound represented by the compound structural formula A-B-C is bonded and fixed to the film surface of the Au thin film 18 through the bond of two sulfur atoms. Then, each of these organic compounds is fixed on the film surface of the Au thin film 18, so that the organic thin film layer 26 made of the organic compound represented by the compound structural formula ABC is formed on the film surface of the Au thin film 18. Are fixed as shown in FIG. That is, the formation of the organic thin film layer 26 on the Au thin film 18 film surface is completed in the first to third steps. The organic thin film layer 26 is the first organic thin film according to the present invention.

In the fourth step following the formation of the organic thin film layer 26 (first organic thin film), an alkaline aqueous solution of hydroxypropyl cellulose (HPC) (4.2 g of HPC and 0.1 mol / liter of sodium hydroxide ( NaO
H) A solution dissolved in 40 ml of an aqueous solution) is prepared to obtain the cellulose hydrogel. With this hydrogel,
The second organic thin film according to the present invention is formed.

In the subsequent fifth step, the sample plate 16 subjected to the above first to third steps is processed into the prepared HPC.
It is immersed in the alkaline aqueous solution for 24 hours and then thoroughly washed with pure water. During this immersion / treatment, the organic thin film layer 2
The carboxyl group of 6 and the unreacted hydroxyl group in HPC are covalently bonded by an ester reaction. Further, in the sixth step, an alkaline solution of bromoacetic acid (bromoacetate 2.
A solution of 7 g dissolved in a 0.1 mol / liter NaOH aqueous solution) was prepared, and thereafter, the sample plate 16 subjected to the fifth step was immersed in the prepared alkaline solution of bromoacetic acid for 16 hours. Process and
After that, thoroughly wash with pure water. During this immersion / treatment, the hydroxyl group which is C of the organic compound represented by the compound structural formula ABC, that is, the hydroxyl group on the film surface of the organic thin film layer 26 is carboxylated to become a carboxyl group. As described above, by the fifth and sixth steps, a second film is formed on the film surface of the organic thin film layer 26.
The organic thin film of is bonded and fixed, and on the Au thin film 18 film surface,
The organic thin film layer 26 and the second organic thin film are formed on the film surface side by bonding and fixing in this order. In other words, the second organic thin film is formed on the Au thin film 18 film surface of the sample plate 16 with the organic thin film layer 26 interposed.

Next, in the seventh step, the second organic thin film on the Au thin film 18 surface is treated with N-hydroxysuccinimide and N- (3-dimethylaminopropyl) -N '.
-A mixed aqueous solution of ethylcarbodiimide hydrochloride (concentrations are 1 mol / liter and 0.1 mol / liter, respectively)
The carboxylic acid groups in the cellulose are activated by contacting with the above for 10 minutes. Then, in the subsequent eighth step, the second organic thin film is brought into contact with a buffer solution of a human albumin antibody (solution in which 10 mg of antibody is dissolved in 10 ml of phosphate buffer) for 10 minutes to activate the above. The human albumin antibody was immobilized on the second organic thin film through the unreacted carboxyl group.

That is, in the eighth step, the organic thin film layer 2
The second organic thin film having six membranes becomes the ligand layer 22 on which the human albumin antibody is immobilized, and the sample plate 16 that can be used for substrate measurement is completed. Then, the completed sample plate 16 is matched with the matching oil 14
A biosensor 10 capable of measuring human albumin, which is a measurement target substrate, if the biosensor 10 is mounted on the prism 12 with a substrate interposed therebetween.
Is completed. In the eighth step, as a post-treatment following immobilization of the human albumin antibody, it was immersed in ethanolamine hydrochloride (pH 8.5, aqueous solution concentration 1 mol / liter) for 5 minutes and then washed with pure water. .

It is also possible to prepare various solutions or hydrogels prepared in the above steps in advance and immerse the sample plate 16 each time. Further, it goes without saying that these preparation steps can be performed in parallel.

A comparison test between the biosensor 10 of the present embodiment and a conventional biosensor (comparative example) will be described below. The biosensor of the comparative example is the Au thin film 18
The structure in which the ligand layer 22 is formed by interposing the organic thin film layer 26 on the film surface is the same as that of the biosensor 10 of this embodiment, but the compound structure of the organic compound forming the organic thin film layer 26 and the hydrogel forming the ligand layer 22 are the same. The type of the biosensor 10 differs from the biosensor 10 of the present embodiment. That is, in the biosensor of this comparative example, the organic thin film layer 26 was formed from 16-mercaptohexadecanol, and the ligand layer 22 was crosslinked with dextran (trade name: T-500, Pharmaci.
aAB) hydrogel. In the biosensor of Comparative Example, 16-mercaptohexadecanol forming the organic thin film layer 26 is an organic compound represented by a compound structural formula X-R-Y, and X in the compound structural formula is an asymmetric or symmetric disulfide, These are diselenide, thiol, and selenol, and R has 10 to 32 carbon atoms.
Is a saturated hydrocarbon chain, and Y is a hydroxyl group, amino group, carboxyl group, or epoxy group. The biosensor 10 of the present example and the biosensor of the comparative example are different in that the hydrogel forming the ligand layer 22 is HPC and crosslinked dextran. However, since both have the same active group (hydroxyl group) for immobilizing a biological substance, this difference does not affect the sensor sensitivity.

In the biosensor of the comparative example having the above structure, the ligand layer and the organic thin film layer were manufactured as follows. In the explanation, the biosensor 10 of the embodiment will be described.
Steps that are the same as or similar to the above will be simplified.

In the first step, a sample plate 16 having an Au thin film 18 deposited and formed thereon is prepared. In the second step, 16-mercaptohexadecanol was added to ethanol /
A solution dissolved in a water mixed solvent is prepared. The concentration, etc.
This is the same as the biosensor 10 of the example. In the third step, as in the case of the biosensor 10, the sample plate 16 is immersed, washed with a solvent, and washed with purified water. In the process of this third step, the sulfur atom of 16-mercaptohexadecanol disulfide is bonded to the gold atom on the Au thin film 18 film surface, and as shown in FIG. An organic thin film layer made of 16-mercaptohexadecanol represented by the structural formula of RY is fixed. This organic thin film layer corresponds to the organic thin film layer 26 in the biosensor 10 of this embodiment.
Next, in the fourth step, an aqueous solution of 0.6 mol / liter of epichlorohydrin and 0.4 mol / liter of diethylene glycol dimethyl ether in NaOH (0.4 mol / liter) was used.
1 / liter). In the subsequent fifth step, the sample plate 16 subjected to the third step is immersed in the above-mentioned aqueous solution for 4 hours. In the fourth and fifth steps, the hydroxyl group of 16-mercaptohexadecanol is converted into an epoxy group.

In the sixth step, an aqueous alkaline solution of crosslinked dextran (3.0 g of crosslinked dextran (10 m
l) 0.1 mol / liter of sodium hydroxide (N
a solution) dissolved in 10 ml of an aqueous solution of aOH) to prepare a hydrogel of cross-linked dextran. In the seventh step, the sample plate 16 is dipped in the alkaline aqueous solution of the prepared crosslinked dextran for 20 hours, and then sufficiently washed with pure water. In the eighth step, as in the case of the biosensor 10, the sample plate 16 is dipped in an alkaline solution of bromacetic acid and then washed with pure water. During this immersion / treatment, Y of 16-mercaptohexadecanol represented by the structural formula of X-R-Y is crosslinked with dextran, and the hydrogel of crosslinked dextran is fixed on the film surface of the organic thin film layer. .

In the ninth step, the hydrogel was treated with N-hydroxysuccinimide and N- (3-dimethylaminopropyl) -N'-, as in the biosensor 10.
It is activated by contact with a mixed aqueous solution of ethylcarbodiimide hydrochloride. In the tenth step, as in the case of the biosensor 10, the human albumin antibody was immobilized on the hydrogel to form a ligand layer. This ligand layer corresponds to the ligand layer 22 in the biosensor 10 of this embodiment.
In addition, in the 10th process, it post-processed similarly to the case of the biosensor 10.

As a comparison test for both sensors of the example and the comparative example, Au of the sample plate 16 of both sensors was used.
A comparison test comparing the film thickness of the organic thin film layer on the film surface of the thin film 18 and a comparison test comparing the sensor sensitivity were performed.
First, a comparison test for film thickness comparison will be described.

In this test, pure water (refractive index: 1.331
When 9) is introduced into the ligand layers of both sensors, the light amount distribution is measured by the CCD image pickup device 28 of each sensor, and an assay solution (1M potassium chloride solution, refractive index: 1.3416) having a predetermined refractive index is measured. The light intensity distribution was also measured when it was introduced into the ligand layer of the sensor. The results are shown in Figure 4.
Is shown in the graph. In the graph, the solid line shows the light amount distribution obtained by the biosensor 10 of the example, and the dotted line shows the light amount distribution obtained by the biosensor of the comparative example. Further, the alternate long and short dash line in the figure is the light amount distribution obtained for pure water with a sensor having a structure in which the Au thin film 18 is simply deposited and formed on the sample plate 16, that is, a sensor having neither the organic thin film layer 26 nor the ligand layer 22. . The vertical axis in FIG. 4 is the relative light amount in order to observe the state of each distribution.

The following was found from FIG. The state of the transition of the distribution compared with the light amount distribution in the sensor having neither the organic thin film layer 26 nor the ligand layer 22 is shown in the biosensor 1 of the example.
0 is less than the biosensor of the comparative example. Further, regarding each sensor, the transition of the light amount distribution in pure water and the light amount distribution in the assay solution is larger in the biosensor 10 of the embodiment, and the resonance angle at which the light amount is at the lowest level is the same as that of the embodiment. The biosensor 10 changed significantly.

In this comparison test, the human albumin antibody immobilized on the ligand layer of each sensor does not cause a biochemical reaction even when the above-mentioned pure water or assay solution is introduced, so that the distribution of the light amount obtained from the sensor changes. That is, the change in the resonance angle corresponds to only the difference between the refractive index of pure water and the refractive index of the assay solution. By the way, in the optical biosensor utilizing the surface plasmon resonance phenomenon on the light reflecting surface, the Au thin film 1
8 Organic thin film layer 2 interposed between the film surface and the ligand layer 22
It is known that the thinner the film thickness of 6, the greater the degree of change in the resonance angle corresponding to the change in the refractive index. Further, it is known that the thinner the film thickness of the organic thin film layer 26, the more similar to the resonance angle in a sensor having no organic thin film layer 26 or the like on the Au thin film 18 film surface.

Therefore, in this comparison test, the same changes in the refractive index were measured for the sensors of the example and the comparative example. Therefore, in combination with the results of the above graph, the biosensor 10 of the example has the organic thin film layer 26. Can be said to be thin.

Next, in a comparison test comparing sensor sensitivities, a human albumin antibody was used as the biological substance immobilized on the ligand layer of the biosensors of Examples and Comparative Examples, and both sensors were used as sensors for detecting human albumin. And
The human albumin measurement solutions prepared at various concentrations shown in the graph of FIG. 5 were successively introduced into both sensors to detect their concentrations. The result is shown in FIG. The vertical axis in FIG. 5 is the sensor output voltage value of the light quantity at the resonance angle, and the horizontal axis is the human albumin concentration (mg / mg / mg) in the measurement solution.
(Deciliter) is the logarithmic axis.

As shown in FIG. 5, in the biosensor 10 of the embodiment, the sensor output has a larger inclination than that of the comparative example, so it can be said that the sensor has high measurement sensitivity. It can be said that this is due to the thinning of the organic thin film layer 26 found in the above-mentioned comparison test of the film thickness comparison.

The embodiments of the present invention have been described above.
The present invention is not limited to such embodiments, and it goes without saying that the present invention can be carried out in various modes without departing from the scope of the present invention.

For example, in the above-mentioned examples, the case where A is a 5-membered disulfide, B is a butyl group and C is a carboxyl group in the compound structural formula A-B-C using thiotic acid is explained. However, it is not limited to this. That is, A of the compound structural formula may be a 5-membered sulfide or a 6-membered or 7-membered sulfide or disulfide. Further, as B of the compound structural formula,
Any saturated hydrocarbon chain having 2 to 8 carbon atoms may be used. However, the saturated hydrocarbon chain of B of this compound structural formula and C of the compound structural formula which is a substituent such as a hydroxyl group, a carboxyl group, an amino group, an aldehyde group, an epoxy group, a vinyl group, a halogen atom, etc. It is determined by the above-mentioned 5-membered, 6-membered or 7-membered sulfide or disulfide which is A in the formula.

The reason why these can be adopted is as follows. The 5-membered ring sulfide or the 6-membered or 7-membered sulfide or disulfide which is A in the compound structural formula A-B-C is a 5-membered ring disulfide and the thioctic acid of the above-mentioned example, and its compound structure This is because they are close to each other and there is no great difference in chemical stability and reactivity. Therefore, the thioctic acid, which is a 5-membered disulfide shown in the examples, can be replaced with a 5-membered sulfide or a 6-membered or 7-membered sulfide or disulfide compound.

The above report (RGNuzzo et al. JACS, 1
983,105,4481-3), a 5-membered ring containing a sulfur atom, 6
The sulfide or disulfide of the member ring is said to strongly adsorb to metals such as gold. Therefore, a sulfide or disulfide compound having at least a 5-membered ring and a sulfide or disulfide having a 6-membered ring can be treated equivalently, and there is no structural reason why a sulfide or disulfide having a 7-membered ring cannot be made equivalent to these.

Further, B in the compound structural formula A-B-C
However, even though the number of carbon atoms is different from that of the butyl group in the examples, there is no great difference in chemical stability or reactivity. Therefore, the butyl group shown in the examples can be substituted with this ethyl group, propyl group, pentyl group and the like.

Similarly, even if C is a hydroxyl group, an amino group, an aldehyde group, an epoxy group, a vinyl group, or a halogen atom in the compound structural formulas A-B-C, the chemical stability and activity of the carboxyl group in the examples, There is no great difference in reactivity. Therefore, the carboxyl group shown in the examples can be substituted with this hydroxyl group, amino group, aldehyde group or the like.

Further, even in the hydrogel of the second organic thin film which becomes the ligand layer 22, compounds of cross-linked dextran, cellulose, starch, agarose, polyacrylamide, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, polyvinyl acetic acid and these are used. Since all the derivatives have a hydroxyl group in common, they can be treated in the same manner. Therefore, the cellulose (HP
C) can be replaced with this crosslinked dextran, starch, agarose, polyacrylamide, or the like.

[0065]

As described above in detail, in the excitation structure of the surface plasmon resonance phenomenon described in any one of claims 1 to 3, a metal thin film formed so that the surface of the light transmitting medium serves as a light reflecting surface. The first organic thin film interposed between the surface and the second organic thin film for immobilizing the biological substance is represented by the compound structural formula A-
As a compound composed of an organic compound represented by BC, this A was a multi-membered ring sulfide or disulfide. Therefore, the first organic thin film is fixed to the surface of the metal thin film through the bonding of the respective organic compounds to the metal thin film by the two sulfur atoms in the multi-membered ring sulfide or disulfide. Therefore, by increasing the number of binding sites per organic compound, the binding of the organic compound to the metal thin film, and thus the binding of the first organic thin film, is strengthened, and the saturated hydrocarbon chain is stabilized to stabilize the first organic thin film. There is no need to align or increase the number of carbon atoms. As a result, claim 1 to claim 3
According to the excitation structure of the surface plasmon resonance phenomenon described in, it is possible to make the first organic thin film interposed on the film surface of the metal thin film uniform and thin, and to increase the number of bonding sites per organic compound. It is possible to suppress the separation of the organic compound and improve the durability.

According to the excitation structure of the surface plasmon resonance phenomenon described in claim 2, the use of a general-purpose compound of a 5- or 7-membered ring sulfide or disulfide eliminates the need for a special synthesizing device or the like, resulting in a significant cost increase. Do not invite.

According to the excitation structure of the surface plasmon resonance phenomenon of claim 3, since the saturated hydrocarbon chain having 2 to 8 carbon atoms is used, the chain length of the organic compound is shortened and the first organic compound is formed. Further thinning of the thin film can be achieved. Moreover, if it is a saturated hydrocarbon chain having 2 to 8 carbon atoms,
Since the raw materials can be easily obtained and synthesized, the cost can be reduced.

In the biosensor according to claim 4, the first organic thin film interposed on the film surface of the metal thin film is made uniform and thinned, and thereby the change of the dielectric constant accompanying the progress of the biochemical reaction is changed. It occurs uniformly in the evanescent region very close to the surface. Therefore, the biosensor according to claim 4 has high measurement sensitivity.

[Brief description of drawings]

FIG. 1 is a schematic diagram schematically showing how an organic compound forming a first organic thin film in the present invention is bonded to a metal thin film.

FIG. 2 is a schematic side view of the biosensor 10 of the embodiment.

FIG. 3 is a CCD image pickup device 28 in the biosensor 10.
The graph which shows the correlation of the incident angle and its light quantity obtained from.

FIG. 4 is a graph for explaining the results of a comparison test between the biosensor 10 of the example and a conventional biosensor.

FIG. 5 is a graph for explaining the result of another contrast test.

FIG. 6 is an explanatory diagram for explaining the configuration of a conventional biosensor and its problems, and is a schematic diagram that schematically shows how organic compounds are bonded to a metal thin film.

[Explanation of symbols]

 10 ... Biosensor 12 ... Prism 14 ... Matching oil 16 ... Sample plate 18 ... Au thin film 20 ... Light reflecting surface 22 ... Ligand layer 24 ... Condensing lens 26 ... Organic thin film layer 28 ... CCD image sensor

Claims (4)

[Claims] 1. A structure which excites a surface plasmon resonance phenomenon by a tunnel effect of light in a light reflection surface that totally reflects incident light, the light transmitting medium forming a transmission path of incident light and reflected light. A metal thin film formed on the surface of the light transmitting medium and having the surface as the light reflecting surface, a first organic thin film formed on the film surface of the metal thin film, and a film of the first organic thin film A second organic thin film formed on the surface, wherein the first organic thin film has a compound structural formula of ABC.
And A in the structural formula of the compound is a multi-membered sulfide or disulfide containing a sulfur atom, B in the structural formula of the compound is a saturated hydrocarbon chain, C is at least one substituent selected from a hydroxyl group, a carboxyl group, an amino group, an aldehyde group, an epoxy group, an active group such as a vinyl group, and a halogen atom, and the first organic thin film is the compound The sulfur atom in the multi-membered ring sulfide or disulfide forming the structural formula A is fixed to the metal thin film by a chemical bond with the metal atom in the metal thin film, and the second organic thin film is a crosslinked dextran, Cellulose, starch, agarose, polyacrylamide, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, polyvinyl chloride Excitation structure of the surface plasmon resonance phenomenon, which is a hydrogel consisting of a derivative of at least one compound selected from acetic acid and / or said compound. 2. The excitation structure for the surface plasmon resonance phenomenon according to claim 1, wherein A in the structural formula of the compound is either a sulfide or a disulfide of a 5- to 7-membered ring. Structure. 3. The excitation structure of the surface plasmon resonance phenomenon according to claim 1, wherein B in the compound structural formula is a saturated hydrocarbon chain having 2 to 8 carbon atoms. Excitation structure of resonance phenomenon. 4. A biosensor using the excitation structure of the surface plasmon resonance phenomenon according to any one of claims 1 to 3, wherein the hydrogel forming the second organic thin film is distinguished from a substrate to be measured. A light irradiating means for fixing and providing a biological substance having a function to cause a biochemical reaction with the substrate, and transmitting light from a light source through the light transmitting medium and condensing and irradiating the light on the light reflecting surface, A biosensor, comprising: a light receiving unit that receives reflected light that is reflected by the light reflecting surface and is emitted from the light transmitting medium to the outside, and that detects the light amount of the reflected light.