JP5056494B2 - Detection surface and method for forming the same - Google Patents

Description

  The present invention relates to a detection surface used in a surface plasmon resonance measurement apparatus, and more particularly to a simple formation method thereof.

2. Description of the Related Art An SPR measuring apparatus that detects the refractive index and refractive index change of a sample by using a surface plasmon resonance (abbreviated as SPR) phenomenon and measures the property of the sample is known.
SPR is an evanescent wave generated when the wave number of an evanescent wave generated by total reflection on the surface of a metal thin film matches the wave number of a surface plasmon wave that is an energy wave generated on the surface of the metal thin film by elementary excitation. It is a phenomenon in which the reflected light intensity of total reflection decreases when used for excitation of surface plasmon waves. The SPR measurement device measures the change in reflected light intensity due to SPR by utilizing the fact that the wave number of the surface plasmon wave changes according to the refractive index of the dielectric when a dielectric is brought into contact with the metal thin film. , And changes in the properties of the sample that cause a change in the refractive index. This device is used in the field of protein research and clinical tests for the measurement of intermolecular interactions using immune reactions, and is a method for detecting intermolecular interactions without the need for labeling substances such as fluorescent substances. There are features.

In an example of the SPR measurement device, when the light emitted from the light source passes through the polarizing plate, only the P-polarized light passes. The P-polarized light is collected by the incident side lens and is incident on the prism. A metal thin film in contact with the object to be measured is provided on the bottom surface of the prism. P-polarized light that has passed through the polarizing plate is incident on the prism at an incident angle θ, and the metal thin film is irradiated to reflect the light from the metal thin film. A change in light intensity is detected by a CCD (Charge Coupled Device) sensor which is a photodetector.
That is, the light emitted from the light source becomes an evanescent wave at the boundary between the prism and the metal thin film, and the wave number is expressed by the equation Kev = Kp · np · sin θ. (Here, Kp is the wave number of incident light, np is the refractive index of the prism, and θ is the incident angle.) On the other hand, a surface plasmon wave is generated on the surface of the metal film, and the wave number is Ksp = (c / w) · It is represented by the formula √ (εn ² / (ε + n ² )). (Where c is the speed of light, w is the angular frequency, ε is the dielectric constant of the metal thin film, and n is the refractive index of the object to be measured.) The incident angle θ at which the wave numbers of the evanescent wave and the surface plasmon wave match. When the evanescent wave is used for excitation of surface plasmon, the amount of light measured as reflected light decreases.

  Since the SPR phenomenon depends on the refractive index of the measurement object in contact with the metal thin film provided on the prism, for example, when the measurement object is water, it can be detected as a curve having a minimum at a certain angle. In addition to measuring the refractive index change due to the concentration change of the antibody, by immobilizing a biomolecule that is a physiologically active substance such as an antibody on a metal thin film, for example, by measuring the refractive index change of the antibody due to binding to an antigen. Analysis of intermolecular interactions of antigen-antibody reactions and quantification of specific substances can be performed. When a specific antigen such as an antigen-antibody reaction is to be studied, it is necessary to immobilize the antibody protein, which is a physiologically active substance, on the metal thin film each time.

Here, the design and preparation of the detection surface (surface on which the object to be measured is formed) on the metal thin film becomes important.
As a method for immobilizing a physiologically active substance on a metal surface, there is a method in which a functional group that binds to a metal is introduced by a chemical bonding method, which is described in Japanese Patent No. 2815120 (Patent Document 1).
In this method, when a metal is immersed in an alkanethiol solution, the thiol forms a specifically strong bond with gold (which is said to be chemisorption), and the molecules are neatly aligned by van der Waals forces of alkyl groups. Since the metal surfaces are coated with an organic film by a chemical bonding method, functional molecules such as physiologically active substances can be immobilized on the metal surface. Various improved inventions are known based on this Patent Document 1.

Using the method described in Japanese Patent No. 2815120 (Patent Document 1), several treatment processes are required to fix a physiologically active substance as a probe having affinity for each substance. According to Patent Document 1, this method needs to go through the following chemical process.
First, to get the basic bond including chemisorption on the gold surface,
(1) Chemosorption of alkanethiol such as 16-mercaptohexadecanol on the gold surface (20 min incubation time)
(2) Epoxy activation by epichlorohydrin treatment of hydroxyl group (4 hour incubation time)
(3) Introduction of dextran with a three-dimensional structure to increase the amount of immobilization (incubation time of 20 hours)
(4) Introduction of carboxymethyl group with bromoacetic acid (incubation for 16 hours)
Process is necessary.
Next, as a technique for immobilizing a physiologically active substance (for example, protein or amino acid) having an amino group on the surface of carboxymethyl-modified dextran produced based on this method, the following technique is disclosed. That is, for example, N-hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) hydrochloric acid can be used to generate a reactive ester function in part of the carboxyl group in carboxymethyl-modified dextran. It is denatured and activated by treatment with an aqueous solution. Residual charges, that is, unreacted carboxyl groups contribute to the concentration of the physiologically active substance on the detection surface. By bringing an aqueous solution of a physiologically active substance (protein or amino acid) containing an amino group into contact with such a detection surface, the physiologically active substance containing an amino group can be covalently bound to the dextran matrix. The hydrogel produced by the method described above exhibits excellent performance as a detection surface of a biosensor because a physiologically active substance containing an amino group can be immobilized three-dimensionally.

However, the hydrogel production method described above is complicated, requires a long production time, and epichlorohydrin may explode when mixed with an acid, and bromoacetic acid is concerned with genotoxicity. There is a concern.
That is, it takes 40 hours or more for the four steps to obtain basic bonding, and it takes time to manufacture this detection surface as a measuring chip.
Furthermore, it is too complicated and time-consuming for a person who conducts immune reaction research, intermolecular interaction measurement, and protein research using a surface plasmon resonance measuring apparatus to carry out on the spot.
The present invention was obtained as a result of studying a detection surface on a simple metal thin film with a small number of processing steps that does not depend on the chemical adsorption method.
Japanese Patent No. 2815120

As a result of intensive studies to solve the above problems, as a method of providing a carboxyl group three-dimensionally on the surface of a substrate such as a metal film of gold, silver, copper, platinum, or aluminum, carboxymethyldextran or the like A carboxyl group can be easily provided by a forming method in which a carboxyl group-containing polymer is mixed with a thickener such as starch, gelatin, or a linear polysaccharide such as agar and physically adsorbed on the metal film to form a film. In addition, even when a physiologically active substance is immobilized and used for measurement, it has been found that a detection surface that can be sufficiently used can be obtained, and the detection surface and the method for forming the same according to the present invention have been completed.
Here, physical adsorption is based on weak adsorption due to van der Waals force without chemical bonding with the metal surface, and also includes an adhesive effect by a thickener.

In the present invention, the method of forming a film by physical adsorption is a method in which a carboxyl group-containing polymer and a thickener are mixed, and then developed on a substrate, and the surface of the metal film is processed smoothly to treat a number of carboxyls on the surface of the metal film. Groups can be placed.
As a method of spreading on the substrate, a method of dropping (dip coating) can be performed more simply, but a spin coating method is preferable as a method having better reproducibility. In addition, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spray coating method, and the like can be used.
Moreover, it is possible to control the carboxyl group content by changing the mixing ratio of the carboxyl group-containing resin and the thickener. The thickener has an effect of arranging the carboxyl groups in a three-dimensional network as well as an effect of making the adsorption to the metal surface stronger.
However, it is possible to dispose a large number of carboxyl groups on the surface of the metal film by using a carboxyl group-containing polymer alone. After dissolving the carboxyl group-containing polymer in a solvent, the above coating method may be applied to the metal substrate. .

As the carboxyl group-containing synthetic polymer used in the present invention, polyacrylic acid, polymethacrylic acid, polymaleic acid and copolymers thereof, methacrylic acid copolymer, acrylic acid copolymer, maleic acid copolymer, partial esterification Examples include maleic acid copolymerization and a polymer having a hydroxyl group to which an acid anhydride is added. Examples of the carboxyl group-containing polysaccharide include carboxymethyl dextran and carboxymethyl starch.
For these substances, commercially available reagents can be used as they are. For example, carboxymethyl dextran is a carboxymethyl dextran sodium salt (trade names CMD, CMD-D40, CMD-L etc.) can be used.
The thickener is preferably a starch, gelatin or a linear polysaccharide such as agar, etc., but if it has an effect to more firmly adsorb to the metal surface and arrange the carboxyl group in a three-dimensional network, It is not limited to the above.

In the basic bonding method to the metal surface thus formed, a carboxymethyl group has already been introduced. Further, in order to fix a physiologically active substance, according to a conventional method,
(1) Activation by adding N-hydroxysuccinimide (NHS) (2) Coupling by adding a physiologically active substance to be immobilized (3) By passing through a process of blocking residual active groups by adding ethanolamine It is.
Furthermore, using the physiologically active substance immobilized in this manner, the analysis of the intermolecular interaction of the antigen-antibody reaction and the quantification of the specific substance can be carried out successfully. The film-formed metal surface is stable to the reagent and solvent used in the analysis operation, and there is no particular problem.

  As described above, according to the detection surface and the method of forming the same of the present invention, when a biomolecule, which is a physiologically active substance such as an antibody, is immobilized on a metal thin film in the SPR measurement device to form a detection surface, the active group on the metal surface The basic bond is a simple method in which a carboxyl group-containing polymer such as carboxymethyl dextran is mixed with a thickener such as starch, gelatin, or a linear polysaccharide such as agar and physically adsorbed onto a gold thin film. Since it can be performed, the expense and time which manufacture the measurement chip | tip of a SPR measuring device can be reduced significantly. In addition, it is safe to use dangerous reagents in manufacturing. Since this detection surface can be performed in a short time using a normal laboratory instrument, a person who conducts immune reaction research, intermolecular interaction measurement, or protein research using an SPR measurement device can apply a metal thin film. The advantage of being able to be used on the spot.

Examples of the present invention will be described below.
In this example, the metal film on the detection surface was formed by depositing chromium of 2 nm or less on a cover glass (catalog code 22-176-01) manufactured by Matsunami Glass Industrial Co. A gold thin film vacuum-deposited to a thickness of 50 nm was used.
Example 1 Example Using Starch as Thickening Agent An example using carboxymethyl dextran and starch as a thickening agent will be described.
<Creation of detection surface>
1. Immediately before use, the gold thin film was preliminarily immersed in 0.1M sodium hydroxide solution and ultrasonically cleaned for 20 minutes, then immersed in pure water and ultrasonically cleaned for 10 minutes 2 to 3 times to adhere to the surface. Remove dirt. As the washing method, a method using an organic solvent such as acetone can also be used.
2. Add 0.01 g starch (from potato) to 10 mL water and heat at 70 ° C. until completely dissolved. To 1 mL of this solution, 1 mL of 0.025% carboxymethyldextran sodium salt (trade name CMD) manufactured by Meisho Sangyo Co., Ltd. is added and mixed well to prepare a mixture.
3. Prepare an SPR substrate of the above 18mm square gold thin film, drop 3μL of the above mixed solution on the gold thin film with a micropipette, and then stretch the surface of the liquid droplet to make it flat and coat by coating method And dried.
4). After drying, the SPR substrate was washed twice with 10 mL of pure water and vacuum dried.
A schematic diagram of the detection surface thus obtained is shown in FIG. The time required for producing such a detection surface by this method is about 1 hour, and it can be produced in a very short time compared to the conventional method. A schematic diagram of the detector surface thus obtained is shown in FIG.

<Immobilization of bioactive substances and their sensorgrams>
Examples of immobilizing proteins on the SPR substrate are shown below.
FIG. 2 is a configuration diagram of an SPR measuring device (manufactured by Toa DKK Corporation) used for immobilization. In FIG. 2, the detection surface is attached to the prism 7 to be the SPR substrate 1. Put PBS buffer in syringe pump 2 and feed at a flow rate of 10 μL / min.
1. Activation treatment: A solution in which an equal amount of 0.1 mol / L N-hydroxysuccinimide (NHS) solution and 0.4 mol / L 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution are mixed from the injector 5 Is injected as a 100 μL sample and developed in a flowing PBS buffer solution. It is left for 15 minutes, during which activation takes place.
2. 100 μL of a solution prepared by dissolving 0.125% bovine albumin (BSA) in pH 4.5 acetate buffer as a protein to be immobilized from the injector 5 is injected and developed in a flowing PBS buffer solution. Allow to stand for 15 minutes, during which time immobilization takes place.
3. Finally, as a masking treatment for preventing nonspecific adsorption, the buffer solution was switched to a 1 mol / L ethanolamine hydrochloride solution adjusted to pH 8.5 and allowed to flow for about 15 minutes. The obtained sensorgram is shown in FIG.

<Measurement of immobilized amount>
The amount of protein immobilized was measured by the SPR measuring device shown in FIG. The SPR substrate 1 is placed on the prism 7, the light from the light source 8 is converged by the lens 9 to irradiate the prism 7, and the light reflected by the SPR substrate 1 is passed through the lens 9 ′ and the polarizing plate 10, and the CCD camera 11. Projected on. The amount of change in the resonance angle of the SPR was calculated from the image captured by the CCD camera 11 by the data processing computer 12. FIG. 3 shows a sensorgram of this immobilization process.
In addition, as a comparative example, the case where the measurement was performed in accordance with the above-mentioned prior patent document 1, a Biacore sensor chip CM-5 (research grade) was attached to an SPR measuring device as a surface to which carboxymethyldextran was bound. The results of operation 3 are shown together in FIG.

The horizontal axis shows the elapsed time of the immobilization treatment, and there is a plateau (plateau state) corresponding to the refractive index of the liquid when the activation liquid is first flowed, and the physiologically active substance bobbin albumin (BSA) is flowed At first, it is shown that bovine albumin enters the immobilized membrane in an excessively concentrated form, and bovine albumin corresponding to the amount of the active group remains immobilized as time passes.
The BSA immobilization angle change was about 7735 RU, which was relatively low compared to the conventional immobilization method of 29150 RU, but about 1/4 of the immobilization amount was obtained, which is practically sufficient. Immobilization amount is obtained.
Thus, it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation simply by mixing carboxymethyl dextran and starch and physically adsorbing them. According to calculation, the amount of immobilization was 7.74 ng / mm ² .

<Measurement of antigen-antibody reaction>
Anti-BSA antibody was measured using a gold thin film on which BSA was immobilized by this method. The measurement procedure is as follows.
1. The SPR substrate 1 on which BSA is immobilized is attached to the SPR measuring device in FIG. 2, PBS buffer is put into the syringe pump 2, and the solution is fed at a flow rate of 50 μL / min.
2. When the angle is stabilized, 100 μL of a solution of 1 μg / mL anti-BSA antibody dissolved in PBS buffer is injected, and the change in angle due to the antigen-antibody reaction is measured.
3. Inject 100 μL of pH 2.0 glycine buffer to dissociate the bound anti-BSA antibody.
4). Similarly, repeat steps 2 and 3 using 2, 5, and 10 μg / mL anti-BSA antibody solution to measure the relationship between antibody concentration and angle change.

  FIG. 4 shows the relationship between antibody concentration and angular change (RU). Moreover, the result of having performed the same measurement by the conventional method was also written together for the comparison. It can be seen that the antibody concentration and the angle change have a good linear relationship, and the antigen-antibody reaction can be measured quantitatively. Moreover, there is no big difference compared with the conventional method, and it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation.

Example 2 Example using gelatin as thickener.
An example using carboxymethyl dextran and gelatin as a thickener will be described.
<Creation of detection surface>
1. Immediately before use, the gold thin film was preliminarily immersed in 0.1M sodium hydroxide solution and ultrasonically cleaned for 20 minutes, then immersed in pure water and ultrasonically cleaned for 10 minutes 2 to 3 times to adhere to the surface. Remove dirt. As the washing method, a method using an organic solvent such as acetone can also be used.
2. Add 0.01 g gelatin to 10 mL water and heat at 90 ° C. until completely dissolved. To 1 mL of this solution, 1 mL of 0.25% carboxymethyldextran sodium salt (trade name CMD) manufactured by Meisho Sangyo Co., Ltd. is added, 8 mL of ethanol is added and mixed well to prepare a mixture.
3. Prepare an 18 mm-square gold thin film SPR substrate and attach it to a spin coater. After dropping 50 μL of the above mixture onto a gold thin film with a micropipette, it was spin-coated at 1000 rpm for 20 seconds and dried.
4). After drying, the SPR substrate was washed twice with 10 mL of pure water and vacuum dried.
<Immobilization of bioactive substances and their sensorgrams>
The same operation as in Example 1 was performed.

<Measurement of immobilized amount>
FIG. 5 shows a sensorgram of this immobilization process.
In addition, as a comparative example in the case where the measurement was performed in accordance with the conventional example, Biacore sensor chip CM-5 (research grade) was attached to an SPR measuring device as a surface to which carboxymethyldextran was bound, and the results are shown in FIG. Also written.
The horizontal axis shows the elapsed time of the immobilization treatment, and there is a plateau (plateau state) corresponding to the refractive index of the liquid when the activation liquid is first flowed, and the physiologically active substance bobbin albumin (BSA) is flowed At first, it is shown that bovine albumin enters the immobilized membrane in an excessively concentrated form, and bovine albumin corresponding to the amount of the active group remains immobilized as time passes.
The change in the BSA fixation angle was about 19630RU, which was lower than the conventional fixation method of 29150RU, but about 2/3 of the fixed amount was obtained.
Thus, it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation only by mixing carboxymethyl dextran and gelatin for physical adsorption. According to the calculation, this immobilization amount is 19.63 ng / mm ² . Further, when gelatin is used, since the gold thin film is coated with gelatin and gelatin itself is a protein, it has an effect of suppressing nonspecific adsorption of other proteins.

Anti-BSA antibody was measured using a gold thin film on which BSA was immobilized by this method. The measurement procedure is as shown in Example 1.
FIG. 6 shows the relationship between antibody concentration and angle change (RU). Moreover, the result of having performed the same measurement by the conventional method was also written together for the comparison. It can be seen that the antibody concentration and the angle change have a good linear relationship, and the antigen-antibody reaction can be measured quantitatively. In addition, the sensitivity is lower than that of the conventional method, but it is not at a level causing practical problems, and it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation.

Example 3 Example of Using Agar as Thickener A description will be given of an example in which agar is used as a linear variety for carboxymethyldextran and thickener.
<Creation of detection surface>
1. Immediately before use, the gold thin film was preliminarily immersed in 0.1M sodium hydroxide solution and ultrasonically cleaned for 20 minutes, then immersed in pure water and ultrasonically cleaned for 10 minutes 2 to 3 times to adhere to the surface. Remove dirt. As the washing method, a method using an organic solvent such as acetone can also be used.
2. Add 0.005 g agar to 10 mL water and heat at 90 ° C. until completely dissolved. To 1 mL of this solution, 1 mL of 0.25% carboxymethyldextran sodium salt (trade name CMD) manufactured by Meisho Sangyo Co., Ltd. is added, 8 mL of ethanol is added and mixed well to prepare a mixture.
3. Prepare an 18 mm-square gold thin film SPR substrate and attach it to a spin coater. After dropping 50 μL of the above mixture onto a gold thin film with a micropipette, it was spin-coated at 1000 rpm for 20 seconds and dried.
4). After drying, the SPR substrate was washed twice with 10 mL of pure water and vacuum dried.
<Immobilization of bioactive substances and their sensorgrams>
The same operation as in Example 1 was performed.

<Measurement of immobilized amount>
FIG. 7 shows a sensorgram of this immobilization process.
In addition, as a comparative example in the case where the measurement was performed in accordance with the conventional example, Biacore sensor chip CM-5 (research grade) was attached to the SPR measuring device as the surface to which carboxymethyldextran was bound, and the results are shown in FIG. Also written.
The horizontal axis shows the elapsed time of the immobilization treatment, and there is a plateau (plateau state) corresponding to the refractive index of the liquid when the activation liquid is first flowed, and the physiologically active substance bobbin albumin (BSA) is flowed At first, it is shown that bovine albumin enters the immobilized membrane in an excessively concentrated form, and bovine albumin corresponding to the amount of the active group remains immobilized as time passes.
The change in BSA fixation angle was about 20140RU, which was lower than the conventional fixation method of 29150RU, but about 2/3 of the fixed amount was obtained.
Thus, it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation simply by mixing carboxymethyl dextran and agar and causing physical adsorption. According to the calculation, this immobilization amount is 20.14 ng / mm ² .

Anti-BSA antibody was measured using a gold thin film on which BSA was immobilized by this method. The measurement procedure is as shown in Example 1.
FIG. 8 shows the relationship between antibody concentration and angular change (RU). Moreover, the result of having performed the same measurement by the conventional method was also written together for the comparison. It can be seen that the antibody concentration and the angle change have a good linear relationship, and the antigen-antibody reaction can be measured quantitatively. In addition, the sensitivity is lower than that of the conventional method, but it is not at a level causing practical problems, and it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation.

Practical example 4 When carboxymethyl dextran is used alone.
Examples using carboxymethyl dextran alone are described.
<Creation of detection surface>
1. Immediately before use, the gold thin film is immersed in 0.1M sodium hydroxide solution and ultrasonically cleaned for 20 minutes, then immersed in pure water and ultrasonically cleaned for 10 minutes 2 to 3 times to adhere to the surface. Remove any dirt. As the washing method, a method using an organic solvent such as acetone can also be used.
2. 0.025 g of carboxymethyl dextran sodium salt (trade name CMD) manufactured by Meisho Sangyo Co., Ltd. was dissolved in 10 mL of ethanol to prepare a 0.25% CMD ethanol solution.
3. Prepare an 18 mm-square gold thin film SPR substrate and attach it to a spin coater. After dropping 50 μL of the above mixture onto a gold thin film with a micropipette, it was spin-coated at 1000 rpm for 20 seconds and dried.
4). After drying, the SPR substrate was washed twice with 10 mL of pure water and vacuum dried.
<Immobilization of bioactive substances and their sensorgrams>
The same operation as in Example 1 was performed.

<Measurement of immobilized amount>
FIG. 9 shows a sensorgram of this immobilization process.
In addition, as a comparative example in the case where the measurement was performed in accordance with the conventional example, a Biacore sensor chip CM-5 (research grade) was attached to the SPR measurement device as a surface to which carboxymethyldextran was bound, and the results are shown in FIG. Also written.
The horizontal axis shows the elapsed time of the immobilization treatment, and there is a plateau (plateau state) corresponding to the refractive index of the liquid when the activation liquid is first flowed, and the physiologically active substance bobbin albumin (BSA) is flowed At first, it is shown that bovine albumin enters the immobilized membrane in an excessively concentrated form, and bovine albumin corresponding to the amount of the active group remains immobilized as time passes.
The change in the BSA fixation angle was about 16700 RU, which was lower than the conventional fixation method of 29150 RU, but an immobilization amount of more than 1/2 was obtained.
Thus, it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation only by physically adsorbing carboxymethyldextran. According to the calculation, this immobilization amount is 16.7 ng / mm ² .

Anti-BSA antibody was measured using a gold thin film on which BSA was immobilized by this method. The measurement procedure is as shown in Example 1.
FIG. 10 shows the relationship between antibody concentration and angular change (RU). Moreover, the result of having performed the same measurement by the conventional method was also written together for the comparison. It can be seen that the antibody concentration and the angle change have a good linear relationship, and the antigen-antibody reaction can be measured quantitatively. In addition, the sensitivity is lower than that of the conventional method, but it is not at a level causing practical problems, and it can be seen that a biosensor excellent in immobilization performance can be produced by a very simple operation.
As described above in detail, according to this method, it is possible to immobilize proteins on a metal thin film by a very simple method as compared with the conventional method, and as a result, desired performance can be obtained. confirmed.

It is a detection surface schematic diagram based on this invention. It is an operation diagram for immobilization based on the present invention. It is a sensorgram of the immobilization process at the time of using starch for a thickener. It is a figure which shows the comparison of the sensitivity with the prior art example at the time of using starch for a thickener. It is a sensorgram of the immobilization process at the time of using gelatin for a thickener. It is a figure which shows the comparison of the sensitivity with the prior art example at the time of using gelatin for a thickener. It is a sensorgram of the immobilization process at the time of using agar for a thickener. It is a figure which shows the comparison of the sensitivity with the prior art example at the time of using agar for a thickener. A sensor gram when the fixing treatment by the carboxyl group-containing polymer alone. It shows a comparison of the sensitivity of the conventional example in the case of a carboxyl group-containing polymer alone.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... SPR board 2 ... Syringe pump 3 ... Buffer liquid 4 ... Switching valve 5 ... Injector 6 ... Waste liquid 7 ... Prism,
DESCRIPTION OF SYMBOLS 8 ... Light source 9 ... Lens 9 '... Lens 10 ... Polarizing plate 11 ... CCD camera 12 ... Computer for data processing

Claims (5)

Gold carboxyl group-containing polymer or a carboxyl group-containing polysaccharide, silver, copper, either directly to the metal surface of the platinum or aluminum, or by a physical adsorption method with thickeners, coalescing been detected surface.   The detection surface according to claim 1, wherein the thickening agent is starch.   The detection surface according to claim 1, wherein the thickener is gelatin.   The detection surface according to claim 1, wherein the thickener is a linear polysaccharide such as agar. Detecting surface carboxyl group-containing polymer or a carboxyl group-containing polysaccharide is gold, silver, copper, either directly to the metal surface of the platinum or aluminum, or by a physical adsorption method with a thickener, characterized by forming a film Forming method.

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