JPH09222395A - Analysis method by surface reinforcing raman scattering measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analysis method with a high sensitivity by amplifying surface reinforcing Raman scattering. SOLUTION: An antibody 1 that is a first specifical connection substance is immobilized to a solid phase 7. An antigen 8 that is a substance to be analyzed is added to the antibody 1 and is connected to the antibody 1. Then, the antibody 2 that is a second specifical connection substance with a Raman active substance generation enzyme 3 is added and is connected to the antigen 8, thus forming a complex of the above three. Then, the antibody 2 that is not related to the formation of the above complex is eliminated by B/F separation. Then, Raman active substance generation substrate 4 is added to the above complex and a Raman active substance (product) 5 is generated by enzyme reaction. Then, the Raman active substance is captured by adsorption using SERS substrate 6 such as silver colloid particle. In this state, excitation light is applied to the above Raman active substance (product) 5 and the surface reinforcing Raman scattered light is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an analytical method used for detecting and quantifying a trace amount of components in the fields of clinical examination, biochemical analysis, quality control of pharmaceuticals, and the like. The present invention relates to an analysis method by the surface enhanced Raman scattering measurement.

[0002]

2. Description of the Related Art An immunoassay utilizing an antigen-antibody reaction (immunoassay) is an analytical method used for detecting and quantifying trace components in the fields of clinical examination, biochemical analysis, quality control of pharmaceuticals, and the like. It is commonly used. Further, as a method of this immunoassay, a method utilizing surface-enhanced Raman scattering measurement has been reported. This method utilizes a phenomenon in which Raman scattering is amplified when a Raman active substance is adsorbed on the surface of metal particles or the like. For example, JP-A-6-174723 and JP-A-7-7-1.
There is a method described in Japanese Patent No. 46295.

[0003]

However, the conventional analysis method has a certain limit in detection sensitivity. For example,
In the method described in JP-A-6-174723, the substance to be analyzed or its specific binding substance is labeled with a Raman active label having a unique Raman scattering pattern, and Raman scattering from this is detected to analyze the substance to be analyzed. A substance is detected or its amount is measured. However, this method utilizes surface-enhanced Raman scattering, but detects only Raman scattering of the Raman-active label itself, and therefore its measurement sensitivity is limited to the Raman scattering detection sensitivity of the label, and higher sensitivity than that. It is theoretically impossible to do. For example,
Rhodamine 6G, which has the highest surface-enhanced Raman scattering intensity among the currently reported, has a detection sensitivity of 10 ^-14.
M (Applied Spectroscopy, 49,6,1985), and crystal violet equivalent to this is 3 × 10 ^-11 M (Anal.
Chem. 58, 1116, 1986). Therefore, even if these dyes are used as Raman activity labels in the method described in JP-A-6-174723, it is not possible to expect a higher detection sensitivity. Further, Japanese Unexamined Patent Publication (Kokai) No. 6-174723 discloses an example in which surface-enhanced Raman scattering is actually applied to an immunoassay, but even with this method, the maximum detection sensitivity is 10 ^-11 M. When the analyte is labeled with the Raman active label, the detection sensitivity tends to be lower than that of the Raman active label in the free state. Therefore, even if the surface-enhanced Raman scattering is applied to the immunoassay, the detection sensitivity becomes lower than that of the Raman-active label itself, and it cannot be said that it is sufficiently compatible with high sensitivity analysis.

In order to solve the above conventional problems, it is an object of the present invention to provide a highly sensitive analysis method by amplifying surface enhanced Raman scattering.

[0005]

In order to achieve the above object, the first analysis method of the present invention comprises a second specific binding substance comprising a first specific binding substance and a Raman active substance-forming enzyme. The binding substance and the analyte are subjected to a binding reaction to form the ternary complex, and the complex and the second specific binding substance not involved in the formation of the complex are separated. A Raman active substance generating substrate is supplied to the complex to react with the enzyme of the second specific binding substance, and then the generated Raman active substance is captured by the surface-enhanced Raman scattering generation substrate, and in this state, the Raman active substance is generated. The active substance is irradiated with excitation light and the generated surface-enhanced Raman scattering is measured.

The second analytical method of the present invention is a Raman active substance which is active when it associates with the second non-active subunit as a competitive substance that competitively binds to the analyte with the specific binding substance. A method for analysis using a substance to be analyzed, which comprises a first non-active subunit that does not associate with the second non-active subunit when the competitive substance binds to the specific binding substance by the production enzyme. The competitive binding reaction of the competitive substance and the analyte with respect to the specific binding substance to form a complex, and then the second non-active subunit is supplied to the competitive substance which is not involved in the formation of the complex. The second non-active subunit is associated with the first non-active subunit of the substance to form an active Raman active substance-generating enzyme, and the Raman active substance-generating group is added to the Raman active substance-generating group. Is supplied and reacted, then the generated Raman active substance is captured by the surface-enhanced Raman scattering generation substrate, and in this state, the Raman active substance is irradiated with excitation light, and the generated surface-enhanced Raman scattering is measured. Take composition.

The third analysis method of the present invention is
The specific binding substance is subjected to a competitive binding reaction between the analyte and a competitor having a Raman active substance-forming enzyme to form a complex, and the complex and the Raman activity not involved in the formation of the complex. After separating the competitive substance having a substance-forming enzyme, a Raman active substance-forming substrate is supplied to the complex to react with the Raman active substance-forming enzyme of the competitive substance, and the produced Raman active substance is surface-enhanced Raman. The Raman active substance is captured by the scattering-occurring substrate, and the Raman active substance is irradiated with excitation light in this state, and the surface-enhanced Raman scattering generated is measured.

That is, the analysis method of the present invention uses a Raman active substance-forming enzyme and a Raman active substance-forming substrate to produce a Raman active substance in an amount proportional (directly proportional or inversely proportional) to the amount of the analyte. is there. By doing so, it becomes possible to remarkably amplify the surface enhanced Raman scattering. As a result, it becomes possible to analyze with a sensitivity far exceeding that of the conventional analysis method.

In the present invention, the first specific binding substance, the second specific binding substance, and the specific binding substance refer to those that selectively bind only to the analyte, for example, an antibody, Examples include oligo DNA, lectin, and receptor. In addition, specific examples of the antibody include anti-insulin antibody, anti-α-fetoprotein antibody, anti-CEA antibody, anti-T ₄ antibody and the like used in clinical tests.

In the present invention, the "Raman activity" means that Raman scattering occurs when irradiated with excitation light, and the "Raman active substance" means the substance having the Raman activity. The "Raman active substance-producing enzyme" refers to an enzyme that converts a specific substrate into a Raman active substance by an enzymatic reaction, and the "Raman active substance-producing substrate" refers to the specific substrate.

The term "surface-enhanced Raman scattering-occurring substrate" refers to a substance having a property of enhancing Raman scattering when a Raman active substance is captured on the surface thereof. And "capture the Raman active substance on the surface-enhanced Raman scattering substrate"
Means that the Raman active substance is brought close to the surface-enhanced Raman scattering generation substrate, and the distance between them is usually 0 to 100 nm, preferably 0 to 50 nm. In addition, as a means of capturing, for example, a method utilizing an adsorption phenomenon, a method of electrically adsorbing by applying a potential, a method of reacting with a surface-enhanced Raman scattering generation substrate substance having a functional group reactively bound to a Raman active substance is used. There is a way.

In the second analytical method of the present invention, as the competitor substance, the analyte substance having the first non-active subunit is replaced with an analyte substance analogue having the first non-active subunit. You may use a thing.

In the third analytical method of the present invention, the competitive substance is an analyte substance having a Raman active substance-forming enzyme for the reason of simplicity of operation and rapidity, and the Raman activity in the competitive substance is The substance-producing enzyme is an enzyme that is inactivated when the analyte substance in the competitor and the specific binding substance are bound to each other, and comprises a complex and the Raman-active substance-forming enzyme that was not involved in the formation of the complex. It is preferable to carry out the reaction of the Raman active substance-forming enzyme without separating it from the competitor.

In the third analytical method of the present invention utilizing the competitive binding reaction, the competing substance having a Raman active substance-forming enzyme is replaced with an analyte substance having a Raman active substance-forming enzyme, It may be an analog of the analyte, which is provided with a Raman active substance-forming enzyme.

In the present invention, the "analyte substance analogue" means a substance whose partial structure is similar to that of the analyte substance and, as a result, binds to the specific binding substance. Further, specific examples of the analyte and the analogue of the analyte include estradiol and estradiol-6-carboxymethyl oxime, testosterone and testosterone-21-hemisuccinate and the like.

For the reason of easiness and quickness of operation, the first specific binding substance or the specific binding substance in the analysis method utilizing the competitive binding reaction is immobilized on a solid phase. preferable.

In order to ensure high measurement accuracy,
The Raman active substance generating enzyme commonly used in all of the present invention, the substrate does not have Raman activity and only the enzymatic reaction product has Raman activity, or the enzyme substrate has Raman activity It is preferably one that can be distinguished from the Raman activity signal of the reaction product. Then, specific preferred examples of the Raman active substance producing enzyme and the Raman active substance producing substrate are as shown below.
These enzymes and substrates, of course, have strong Raman scattering signals, but since the Raman active substance produced is a colored substance, a resonance Raman effect can also be expected. It becomes possible to measure.

First, the Raman active substance producing enzyme is peroxidase, and the Raman active substance producing substrate is 2,2 '.
-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) [2,2-Azino-bis
(3-ethylbenzthiazoline-6-
sulphonic acid)], o-phenylenediamine (OP
D)], 3,3 ', 5,5'-Tetramethylbenzidine [3,3', 5,5'-Tetramethylbenzyl
zine], o-dianisidine [o-Dianis
idine], 5-amino salicylic acid [5-
Aminosalicyclic acid], 3,3 '
-Diaminobenzene [3,3'-Diaminoben
ze], 3-amino-9-ethylcarbazole [3-A
[mino-9-ethylcarbazole], 4-
Chloro-1-naphthol [4-Chloro-1-na
It is preferably at least one selected from the group consisting of [phthol].

The Raman active substance producing enzyme is alkaline phosphatase, and the Raman active substance producing substrate is p-nitrophenyl phosphate [p-Nitr].
ophenyl phosphate], 5-bromo-
4-Chloro-3-indolyl phosphate [5-Br
omo-4-chloro-3-indolyl ph
osphate], Fast red phosphate [Fast red phosphate], 4-methylambelliferyl phosphate [4-Methylu]
It is preferably at least one selected from the group consisting of mbelliferyl phosphate].

The Raman active substance producing enzyme is glucosidase, and the Raman active substance producing substrate is p-nitrophenyl glucoside [p-Nitropheny.
lglucoside] and 4-methylambelliferyl glucoside [4-Methylumbellif
It is preferably at least one of [erylglucoside].

The Raman active substance producing enzyme is galactosidase, and the Raman active substance producing substrate is p-nitrophenylgalactoside [p-Nitrophen].
ylgalactoside] and 4-methylambelliferyl galactoside [4-Methylumbe
It is preferably at least one of [lliferylgalactoside].

The Raman active substance producing enzyme is sulfatase, and the Raman active substance producing substrate is p-nitrophenylsulfate [p-Nitropheny.
lSulfate] and 4-methylambelliferyl sulfate [4-Methylumbellifer
yl sulphate].

Further, the Raman active substance producing enzyme is urease, and the Raman active substance producing substrate is urea and bromocresol purple [Bromocresol].
It is preferable that it is at least one.

The surface-enhanced Raman scattering-occurring substrate commonly used in the present invention is a metal colloid, a metal film, a metal foil, an electrode, or a conductor because a conductive substance easily causes surface-enhanced Raman scattering. And at least one selected from the group consisting of semiconductors. Specific examples include silver colloid, gold colloid, silver electrode, silver film, CaP and the like.

[0025]

Next, the present invention will be specifically described.

FIG. 1 shows an example of the first analysis method of the present invention.
It will be described based on. In this example, an antigen-antibody reaction is used.

That is, first, the antibody 1 which is the first specific binding substance is immobilized on the solid phase 7. Examples of the solid phase 7 include polystyrene, latex, sepharose, polyacrylamide, and amino group-introduced latex. Moreover, examples of the fixing method include a physical adsorption method and a chemical bonding method. Then, the antigen 8 which is a substance to be analyzed is added to the antibody 1 to bind to the antibody 1. Then, the antibody 2 which is the second specific binding substance is added to bind to the antigen 8. This antibody 2 is labeled with a Raman active substance-forming enzyme 3. In this way, antibody 1
And the antibody 2 is bound in a sandwich form with the substance to be analyzed (antigen) 8 sandwiched therebetween, and the above three forms a complex. The analyte (antigen) 8 and the antibody 2 may be added at the same time. Then, the antibody 2 not involved in the formation of the complex is removed by B / F separation (Bound / Free separation). As a method for this separation, a conventionally known method applied to an immunoassay can be used, and examples thereof include washing with a buffer solution and a method utilizing a chromatographic phenomenon. Then, the Raman active substance-generating substrate 4 is added to the complex to generate a Raman active substance (product) 5 by an enzymatic reaction. Then, this Raman active substance is captured by the surface-enhanced Raman scattering generation substrate (SERS-converted substrate) 6. This capture can be performed by the adsorption method described above or the like. Then, in this state, the Raman active substance (product) 5 is irradiated with excitation light, and the generated surface-enhanced Raman scattered light is measured. Since this surface-enhanced Raman scattering depends on the amount of the generated Raman active substance,
It is possible to increase the strength by increasing the amount of Raman active substance. Therefore, in the analysis method of the present invention, it is possible to improve the detection sensitivity in proportion to the amount of the substance to be analyzed, and it is possible to perform high-sensitivity analysis at a level that cannot be achieved by the conventional method.

For the irradiation of the excitation light and the measurement of the surface-enhanced Raman scattering, a general Raman scattering measuring device can be used. The measurement conditions are appropriately determined depending on the type and addition amount of the enzyme or substrate to be used, the concentration of the substance to be analyzed, and the like. Normally, the intensity of the argon ion laser is 50 mW to 200 mW, and the higher the intensity, the better.
Further, it is desirable that the Raman scattering generation substrate is in a large excess over the Raman active substance produced by the enzyme.

Next, the second analysis method of the present invention is
This method uses a Raman active substance-forming enzyme having a subunit structure and utilizes a competitive binding reaction.

A specific example of this method is shown in FIG. This specific example utilizes an antigen-antibody reaction as in the first method of the present invention described above. In the figure, the operation flow is from left to right. As shown, first,
An analyte (antigen) 8 and an analyte 8 having the first non-active subunit 3a which is a competitor thereof are prepared, and an antibody 1 which is a specific binding substance is supplied thereto. Then, when there are many competitors, the competitor and the antibody 1 mainly form a complex, and when there are many analytes 8, the analyte 8 and the antibody 1 mainly form a complex. The figure shows the case where a complex is formed and the case where a complex is not formed in a competitive substance. When the second non-active subunit 3b is supplied to these, the second non-active subunit 3b is associated and activated only with the first non-active subunit in the competitor that does not form a complex. Raman active substance-forming enzyme 3 is formed. At this time, the first non-active subunit 3a in the complex undergoes a steric structural change and is unable to associate with the second non-active subunit 3b. In this state, when the substrate for producing the Raman active substance is supplied, the Raman active substance is produced by the enzymatic reaction according to the amount of the competing substance not involved in the complex formation. Then, the Raman active substance is captured by the surface-enhanced Raman scattering generation substrate, and in this state, the Raman active substance is irradiated with excitation light, and the surface-enhanced Raman scattering generated is measured.

When the competitive binding reaction is used in this way, the amount of the substance to be analyzed and the Raman scattering intensity have a direct proportional relationship. As described above, when the amount of the analyte is larger than that of the competitor, the analyte mainly binds to the specific binding substance, and the intensity of Raman scattering generated by increasing the number of competitors not involved in complex formation increases. On the contrary, when the amount of the analyte is smaller than that of the competitor, mainly the competitor binds to the specific binding substance, so that the number of competitors not involved in the complex formation decreases and the Raman scattering intensity decreases. .

Further, in this analysis method, the Raman active substance-forming enzyme having a subunit structure is used, and the separation operation between the complex and the substance not involved in the formation of the complex can be omitted. is there.

The Raman active substance-forming enzyme having a subunit structure used in this analysis method is, for example, β-galactosidase.

Next, the third analysis method of the present invention is
This is a method utilizing a competitive binding reaction as in the above-mentioned second method of the present invention. In this method, a specific binding substance is subjected to a competitive binding reaction between an analyte and a competitor having a Raman active substance-forming enzyme to form a complex, which is involved in the formation of this complex and the complex. After separating from the competitor substance having the Raman active substance-forming enzyme which was not present, the Raman active substance-forming substrate is supplied to the complex to react with the Raman active substance-forming enzyme, and the generated Raman active substance is surface-treated. This is a method of capturing surface-enhanced Raman scattering, and then irradiating the Raman active substance with excitation light in this state to measure the surface-enhanced Raman scattering generated.

In this method, contrary to the above-mentioned second method of the present invention, the amount of the substance to be analyzed and the Raman scattering intensity have an inversely proportional relationship. That is, when the amount of the substance to be analyzed is larger than that of the competitor, the substance to be analyzed mainly binds to the specific binding substance, and the generated Raman scattering intensity becomes weak. On the contrary, when the amount of the analyte is smaller than that of the competitor, the Raman scattering intensity is increased mainly because the competitor binds to the specific binding substance.

Then, in the analysis method utilizing this competitive binding method, in the analyte substance having the Raman active substance-forming enzyme as the Raman active substance-forming enzyme, deactivation occurs when the analyte substance binds to the specific binding substance. As described above, it is possible to omit the separation operation of the complex and the competitive substance not involved in the complex formation by using the enzyme. Examples of such an enzyme include β-
Galactosidase is an example.

Next, examples will be described. In addition,
"SERS" referred to below means surface-enhanced Raman scattering.

[0038]

Example 1 (Preparation of silver colloid solution) 50 ml of 1 mM silver nitrate and 2 m
A silver colloid solution was prepared by mixing 150 ml of M sodium borohydride with stirring under ice cooling and reducing the mixture. The maximum absorption wavelength was 390 nm. In addition,
Beakers and other equipment used were washed with purified water or distilled water, and care was taken to avoid contamination with salts as much as possible.

(Enzyme reaction conditions) 10 ^-6 mol / mlo-
10 ml of phenylenediamine (100 mM phosphate buffer solution
(dissolved in pH 5), 13.6 μl of 30% hydrogen peroxide was added. 5 μg / ml peroxidase (POD,
10 μl of Oriental Yeast Co., Ltd. was added, and the mixture was added at 30 ° C. for 2
When the enzyme reaction was carried out for 0 minutes, an azo compound having a maximum absorption wavelength of 420 nm was produced. The production reaction of this azo compound is shown in FIG.

(SERS generation condition) The silver colloid solution and the enzyme reaction solution were mixed at a volume ratio of 9: 1, and SER was generated by an argon laser (excitation wavelength 514 nm, output 50 mW).
S measurement (wave number 1442 cm ⁻¹ ) was performed. A measuring device manufactured by Princeton was used as the measuring device at this time. The SERS spectrum is shown in FIG. It was previously confirmed that SERS did not occur in each reagent before the enzymatic reaction.
That is, the SERS of 0.136% hydrogen peroxide, 0.1% POD, and 10 ⁻⁶ mol / ml o-phenylenediamine at the same concentrations as in the enzyme reaction were measured. As a result, as shown in the graphs of FIGS. 5, 6 and 7, the occurrence of SERS was not observed in any of them.

(SERS-enzyme immunoassay) 10 μg / ml goat anti-mouse I was added to an immunoassay microplate.
After adding 100 μl of GG (polyclonal antibody, MBL) and incubating at room temperature for 2 hours, 0.5%
Blocking with BSA (bovine serum albumin) 1 at 4 ℃
I went for 2 hours. On this sensitized plate, 10, 5, 2.
5, 1.25, 0.625, 0.315, 0.1575n
100 μl of g / ml mouse IgG (manufactured by Dako) was added, and the mixture was reacted at room temperature for 1 hour, separated by B / F, and washed. To this, 100 μl of an anti-mouse IgG-POD labeled antibody (manufactured by Dako) was added and reacted for 1 hour, and then B
/ F was separated and washed. Next, 00 μl of 12 mMo-orthophenylenediamine-0.136% hydrogen peroxide dissolved in 0.1 M phosphate citrate buffer (pH 5.0) was added and reacted for 20 minutes. SERS measurement was performed on the reaction solution in the same manner as the SERS generation condition.
When the relationship between the intensity and the concentration of mouse IgG (antigen) was plotted, the calibration curve shown in FIG. 8 was obtained.

According to this, the detection limit is 0.5 ng /
It is less than ml, and it can be seen that the sensitivity is higher than that of the conventional analysis method.

[0043]

INDUSTRIAL APPLICABILITY As described above, the analysis method of the present invention is an analysis method capable of high-level detection and measurement, which cannot be achieved by the conventional analysis method utilizing surface-enhanced Raman scattering. In addition, the operation is simple and easy to carry out. Therefore, by applying the analysis method of the present invention, the reliability of the inspection and analysis will be improved in the fields of clinical examination, biochemical analysis, quality control of pharmaceuticals, and the like.

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating a concept in an example of the present invention.

FIG. 2 is a schematic diagram illustrating the concept of another example of the present invention.

FIG. 3 is a diagram showing an oxidative condensation reaction of o-phenylenediamine and hydrogen peroxide by POD in one example of the present invention.

FIG. 4 SERS of enzyme reaction product in the above-mentioned Example
It is a spectrum diagram.

FIG. 5 is a SERS spectrum diagram of a hydrogen peroxide solution in the example.

FIG. 6 SER of peroxidase in the above example
It is an S spectrum diagram.

FIG. 7 is a SERS spectrum diagram of o-phenylenediamine in the example.

FIG. 8 is a graph showing the relationship between mouse IgG concentration and SERS intensity in the SERS-enzyme immunoassay in the above Example.

[Explanation of symbols]

 1 antibody 2 antibody 3 enzyme 4 enzyme substrate 5 product 6 SERS-ized substrate 7 solid phase 8 antigen

Claims (16)

[Claims] 1. A first specific binding substance, a second specific binding substance provided with a Raman active substance-forming enzyme, and an analyte are allowed to react with each other to form a complex of the three parties. After separating the complex from the second specific binding substance that is not involved in the formation of the complex, a Raman active substance-forming substrate is supplied to the complex to remove the second specific binding substance. A method for analysis in which a surface-enhanced Raman scattering substance is reacted with the enzyme, the surface-enhanced Raman-scattering substrate is captured, the excitation light is irradiated to the Raman-active substance in this state, and the generated surface-enhanced Raman scattering is measured. 2. The analysis method according to claim 1, wherein the first specific binding substance is immobilized on a solid phase. 3. A Raman active substance-forming enzyme in an active form is produced as a competitive substance for causing a competitive binding reaction with a substance to be analyzed for a specific binding substance with a second non-active subunit, and the competitive substance is A method of analysis using an analyte comprising a first inactive subunit that does not associate with the second inactive subunit when bound to the specific binding substance, wherein The analyte is subjected to a competitive binding reaction to form a complex, and then the second non-active subunit is supplied to the first non-active subunit of the competitor not involved in the formation of the complex. The second non-active subunit is allowed to associate with each other to form an active Raman active substance-generating enzyme, and the Raman active substance-generating substrate is supplied to react with the Raman active substance-generating enzyme, and then the enzyme is generated. An analysis method in which a Raman active substance is captured by a surface-enhanced Raman scattering-occurring substrate, and in this state, the Raman active substance is irradiated with excitation light to measure the surface-enhanced Raman scattering generated. 4. The analysis method according to claim 3, wherein as the competitor, an analyte analog having a first non-active subunit is used instead of the analyte having the first non-active subunit. . 5. A specific binding substance is allowed to undergo a competitive binding reaction between an analyte and a competitor having a Raman active substance-forming enzyme to form a complex, which is involved in the formation of this complex and the complex. Was separated from the competitor with the Raman active substance-forming enzyme that was not present, and then a Raman active substance-generating substrate was supplied to the complex to react with the Raman active substance-generating enzyme of the competitive substance to generate Raman activity. An analysis method in which a substance is captured on a surface-enhanced Raman scattering-occurring substrate, and the Raman active substance is irradiated with excitation light in this state to measure the surface-enhanced Raman scattering generated. 6. The competing substance is an analyte containing a Raman active substance-forming enzyme, and the Raman active substance producing enzyme in the competitor binds to the analyte in the competitor and a specific binding substance. 6. The reaction of the Raman active substance-forming enzyme is carried out without separating the complex and the competitive substance having the Raman active substance-generating enzyme that was not involved in the formation of the complex. Analytical method described in. 7. The analysis method according to claim 6, wherein, as the competitor, an analyte analog having a Raman active substance-forming enzyme is used in place of the analyte having a Raman active substance-producing enzyme. 8. The analysis method according to claim 5, wherein the specific binding substance is immobilized on a solid phase. 9. A Raman active substance-forming enzyme whose substrate does not have Raman activity and only the enzymatic reaction product has Raman activity, or Raman which is an enzymatic reaction product whose enzyme substrate has Raman activity. The analysis method according to any one of claims 1 to 8, which is distinguishable from an activity signal. 10. The Raman active substance producing enzyme is peroxidase, and the Raman active substance producing substrate is 2,2′-
Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), o-phenylenediamine, 3,
3 ', 5,5'-tetramethylbenzidine, o-dianisidine, 5-aminosalicylic acid, 3,3'-
The analysis according to any one of claims 1 to 9, which is at least one Raman active substance-forming substrate selected from the group consisting of diaminobenzene, 3-amino-9-ethylcarbazole, and 4-chloro-1-naphthol. Method. 11. The Raman active substance producing enzyme is alkaline phosphatase, and the Raman active substance producing substrate is
p-nitrophenyl phosphate, 5-bromo-4-
10. At least one Raman active substance-producing substrate selected from the group consisting of chloro-3-indolyl phosphate, fast red phosphate, and 4-methylambelliferyl phosphate. Analytical method described. 12. The Raman active substance producing enzyme is glucosidase, and the Raman active substance producing substrate is at least one Raman active substance producing substrate of p-nitrophenyl-glucoside and 4-methylambelliferyl glucoside. The analysis method according to any one of claims 1 to 9. 13. The Raman active substance producing enzyme is galactosidase, and the Raman active substance producing substrate is at least one Raman active substance producing substrate of p-nitrophenyl-galactoside and 4-methylambelliferylgalactoside. The analysis method according to any one of claims 1 to 9. 14. The Raman active substance producing enzyme is sulfatase, and the Raman active substance producing substrate is at least one Raman active substance producing substrate of p-nitrophenylsulfate and 4-methylambelliferylsulfate. The analysis method according to any one of claims 1 to 9. 15. The Raman active substance producing enzyme is urease, and the Raman active substance producing substrate is at least one Raman active substance producing substrate of urea and bromocresol purple. Analytical method described. 16. The surface-enhanced Raman scattering-occurring substrate is at least one selected from the group consisting of metal colloids, metal films, metal foils, electrodes, conductors and semiconductors. Analytical method described in.