JP7104911B2 - Target substance detection method and waveguide mode sensor - Google Patents

Description

The present invention provides a target substance detection method that simplifies the process for capturing a target substance with avidin or the like when detecting a target substance using a waveguide mode sensor, and a waveguide suitable for implementing the target substance detection method. Regarding mode sensors.

A waveguide mode sensor is used as a sensor for detecting a minute substance present in a solution as a target substance (for example, see Non-Patent Documents 1 to 3).
The sensor chip responsible for detecting the target substance in the waveguide mode sensor includes a first layer formed of any one of a metal material, a semiconductor material, and a first dielectric material on a light transmissive substrate; A second layer made of a second dielectric material is laminated in this order.
When such a sensor chip is irradiated with the light at a specific incident angle from the rear surface side (the light-transmitting substrate side) while satisfying the conditions for total reflection, light of a specific wavelength is projected onto the first layer and the second layer. is coupled with a waveguide mode (also called an optical waveguide mode, waveguide mode, optical waveguide mode, leaky mode, etc.) propagating in the layer, and the waveguide mode is excited.

As a method of performing sensing by the waveguide mode sensor, there is a first method of detecting reflected light of the light irradiated to the sensor chip.
In this first method, when incident light of a specific wavelength is incident on the waveguide mode sensor, the intensity of the reflected light changes at a specific incident angle at which the waveguide mode is excited, or It takes advantage of the fact that when incident light with a spectral width at an angle is incident on the guided mode sensor, the intensity of the reflected light changes at the particular wavelength at which the guided mode is excited.
The first method utilizes the fact that the reflected light intensity near the incident angle or near the specific wavelength at which the waveguide mode is excited varies depending on the complex refractive index near the surface of the sensor chip.
That is, when the target substance is adsorbed to or approaches the surface of the sensor chip and the complex refractive index changes, the reflected light intensity near the incident angle or the specific wavelength changes. The target substance can be detected as a characteristic change of the reflected light.
Specifically, the adsorption of the target substance is measured at the position and depth of a dip that shows a sharp decrease in reflected light intensity at a specific incident angle when light of a specific wavelength is incident, or at a specific incident angle. Adsorption of the target substance on the surface of the sensor chip can be detected by using a detector to detect the position and depth of the dip that shows a sharp decrease in reflected light intensity at a specific wavelength when light with broad wavelength characteristics is incident. can be detected.
The waveguide mode excitation condition is sensitive to changes in the imaginary part of the complex refractive index on the surface of the sensor chip, that is, changes in absorbance. , the waveguide mode sensor can be expected to detect the target substance with high sensitivity.

As another method of performing sensing by the waveguide mode sensor, there is a second method of detecting fluorescence or scattered light associated with the presence of the target substance emitted based on the light illuminating the sensor chip.
In this second method, the light is irradiated from the back side of the sensor chip at a specific incident angle while satisfying the conditions for total reflection, and when the waveguide mode is excited, an enhanced electric field is formed near the surface of the sensor chip. take advantage of what is available.
That is, when the enhanced electric field is formed, the target substance itself or the labeling substance that labels the target substance introduced onto the surface of the sensor chip emits fluorescence or scattered light. , it becomes possible to detect the presence of the target substance.

By the way, when the waveguide mode sensor detects a specific biological substance or the like as the target substance, after immobilizing an antibody that specifically captures the target substance on the surface of the sensor chip, the antibody The target substance (antigen) is detected by capturing the target substance (antigen) through an antigen-antibody reaction.
In this conventional method, first, after the surface of the sensor chip is modified with a compound having a biotin site, streptavidin or a reagent obtained by binding the antibody to avidin is introduced onto the surface of the sensor chip, and the biotin site is By binding the streptavidin or the like, a pretreatment is performed to immobilize the antibody on the surface of the sensor chip via the compound having a biotin site and the streptavidin or the like.
Next, a sample liquid is introduced onto the surface of the sensor chip, and a capturing process is performed to capture the target substance (antigen) by the antigen-antibody reaction with respect to the antibody.
Next, detection of the target substance contained in the sample liquid through comparison of each sensing state of the waveguide mode sensor in a state where the antibody captures the target substance and a state where the antibody does not capture the target substance. Take action.

However, if the reagent and the subject liquid are handled separately, it is necessary to mix the reagent and the subject liquid for the antigen-antibody reaction on the surface of the sensor chip where the space is limited. Therefore, the work before performing the detection process becomes complicated. In addition, in addition to the capture treatment for the antigen-antibody reaction, the pretreatment for binding the compound having a biotin site to the antibody-attached streptavidin or the like is necessary. work becomes step-by-step, further complicating the work before performing the detection process.
Furthermore, the conjugate in which the antibody-attached streptavidin or the like that is unbound to the compound having a biotin site is bound to the antigen in the capture treatment cannot be distinguished from contaminants in the detection treatment. and the capture treatment, a washing step is required to wash the compound having the biotin site and the unbound streptavidin and the like with the antibody.
Therefore, the conventional method has the problems that the operation is complicated, the number of steps required for detection increases, and the detection of the target substance takes a long time.

Nanotechnology Vol. 19, 095503 (2008) Optics Express Vol. 18, No. 15, 15732 (2010) Optics Express Vol. 25, No. 21, 26011 (2017)

The present invention solves the aforementioned problems in the prior art, and provides a target substance detection method capable of simplifying the target substance detection operation using a waveguide mode sensor, and the waveguide mode suitable for implementing the target substance detection method. An object is to provide a sensor.

＜２＞ 試薬が、導波モードセンサでセンシング可能な標識物質と、前記標識物質と結合されるとともに標的物質と結合して捕捉可能とされる第２の標的物質捕捉物質とを更に含む前記＜１＞に記載の標的物質検出方法。
＜３＞ 第１の標的物質捕捉物質及び第２の標的物質捕捉物質が、標的物質に対する抗体及びアプタマーのいずれかである前記＜２＞に記載の標的物質検出方法。
＜４＞ 標識物質が、可視光領域において光吸収性を有する色素及びナノ粒子、蛍光を発する色素及び微粒子、並びに、散乱光を発する微粒子のいずれかである前記＜２＞から＜３＞のいずれかに記載の標的物質検出方法。
＜５＞ 導波モードセンサによるセンシングが、センサチップの裏面側から全反射条件で照射された光の反射光を検知して実施される前記＜１＞から＜４＞のいずれかに記載の標的物質検出方法。
＜６＞ 標的物質検出工程が、基準状態と測定状態とでそれぞれ検知された反射光の反射光スペクトルにおけるディップ底部の深さを比較して実施される前記＜５＞に記載の標的物質検出方法。
＜７＞ 導波モードセンサによるセンシングが、センサチップに対し裏面側から全反射条件で光を照射することに基づいて発せられる標的物質の存在に伴う蛍光又は散乱光を検知して実施される前記＜１＞から＜４＞のいずれかに記載の標的物質検出方法。
＜８＞ シラン化合物にビオチンが付加されたビオチニル化シランカップリング剤により表面修飾されたセンサチップが配され、前記ビオチニル化シランカップリング剤が、下記一般式（１）に示す化合物であることを特徴とする導波モードセンサ。
ただし、前記一般式（１）中、Ｒ ^１ は、炭素数が２～１０のアルキレン基を示し、Ｒ ^２ は、炭素数が２～１５のアルキレン基を示す。

Means for solving the above problems are as follows. Namely
<1> A biotin immobilization step of immobilizing biotin on the surface of a sensor chip of a waveguide mode sensor, an analyte for which the presence of a target substance to be detected is verified, and at least one of streptavidin and avidin. and a reagent containing a first target substance-capturing substance that is bound to the biotin-conjugate and that binds to the target substance to be captured by the biotin-conjugate. a mixed solution introducing step of introducing the mixed solution onto the surface of the sensor chip on which the biotin is immobilized; A sensing state of the waveguide mode sensor when a reference solution, which is either the mixed solution in which the contents of the reagent and the target substance are controlled, is introduced onto the surface of the sensor chip on which biotin is immobilized. is a reference state, and the sensing state of the waveguide mode sensor when the mixed liquid is introduced in the mixed liquid introduction step is set as a measurement state, and the target substance is detected by comparing the reference state and the measurement state. and a target substance detection step, wherein the biotin immobilization step is to bring a solution of a biotinylated silane coupling agent in which biotin is added to a silane compound into contact with the surface of the sensor chip, and then the sensor chip is A method for detecting a target substance, which is a step of drying , wherein the biotinylated silane coupling agent is a compound represented by the following general formula (1) .
However, in the general formula (1), R ¹ represents an alkylene group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 2 to 15 carbon atoms.

<2> The reagent further includes a labeling substance that can be sensed by the waveguide mode sensor, and a second target substance-capturing substance that is bound to the labeling substance and binds to the target substance to be captured by the <1>, the method for detecting a target substance.
<3> The target substance detection method according to <2> above, wherein the first target substance-capturing substance and the second target substance-capturing substance are either an antibody or an aptamer against the target substance.
<4> Any of <2> to <3> above, wherein the labeling substance is any one of dyes and nanoparticles that absorb light in the visible light region, dyes and fine particles that emit fluorescence, and fine particles that emit scattered light. The method for detecting a target substance according to .
< 5 > The target according to any one of <1> to < 4 >, wherein sensing by the waveguide mode sensor is performed by detecting reflected light of light irradiated from the back side of the sensor chip under conditions of total internal reflection. Substance detection method.
< 6 > The target substance detection method according to < 5 > above, wherein the target substance detection step is performed by comparing the depth of the dip bottom in the reflected light spectrum of the reflected light detected in the reference state and the measurement state. .
< 7 > Sensing by the waveguide mode sensor is performed by detecting fluorescence or scattered light associated with the presence of the target substance, which is emitted based on irradiation of light from the rear surface side of the sensor chip under conditions of total internal reflection. The target substance detection method according to any one of <1> to < 4 >.
< 8 > A sensor chip whose surface is modified with a biotinylated silane coupling agent in which biotin is added to a silane compound is arranged, and the biotinylated silane coupling agent is a compound represented by the following general formula (1): A waveguide mode sensor characterized by:
However, in the general formula (1), R ¹ represents an alkylene group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 2 to 15 carbon atoms.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to solve the above-mentioned problems in the prior art, and it is suitable for implementing a target substance detection method capable of simplifying the target substance detection operation using a waveguide mode sensor, and the target substance detection method. It is possible to provide the above-mentioned waveguide mode sensor.

FIG. 10 is a diagram showing an example of detection of a dip caused by a sudden decrease in reflected light intensity at a specific wavelength when light having broad wavelength characteristics is incident at a specific incident angle; It is an explanatory view for explaining a 1st embodiment. FIG. 4 is an explanatory diagram showing an outline of a biotin immobilization step; It is explanatory drawing which shows the outline|summary of a liquid mixture preparation process. It is explanatory drawing which shows the outline|summary of a liquid mixture introduction process. It is an explanatory view for explaining a 2nd embodiment. FIG. 3 shows measurement results at dip positions of reflected light spectra for negative control, positive control, and CRP antigen 100 pM and 200 pM samples. FIG. 10 is a diagram showing measurement results at dip positions of reflected light spectra for negative control and CRP antigen 10 pM and 50 pM samples. FIG. 10 shows measurement results at dip positions of reflected light spectra for a negative control, a positive control, and a sample containing 300 pM of CRP antigen.

(Basic configuration of waveguide mode sensor)
First, the basic configuration of a waveguide mode sensor to which the target substance detection method of the present invention is applied will be described, and then the target substance detection method will be described based on the basic configuration of the waveguide mode sensor.

The waveguide mode sensor includes a sensor chip, a liquid holding section, a light irradiation section, and a light detection section.

<Sensor chip>
The sensor chip includes a light-transmitting substrate and a waveguide mode excitation layer, and a waveguide mode is excited in the waveguide mode excitation layer when light is irradiated from the light-transmitting substrate side under conditions of total reflection. configured to be

-Light transmissive substrate-
The light-transmitting substrate is a substrate made of a known glass material or plastic material.
In this specification, the term "light transmissive" means that the visible light transmittance is 0.5% or more.

The glass material, which is the material for forming the transparent substrate, is not particularly limited. Silica glass is preferable from the viewpoint of obtaining a substrate. Examples of the silica glass include silica glass itself formed of silicon dioxide (SiO ₂ ) as a single component, silica glass containing impurities such as fused silica glass, boron silica glass, fluorine-doped silica glass, and germanium-doped silica. It includes glass obtained by adding an additive to silica glass such as glass, and includes, for example, BK7 glass and Pyrex glass (registered trademark).
As the plastic material, general transparent plastic materials such as polystyrene, polycarbonate, acrylic, polyester, and COP can be used.

- Guided mode excitation layer -
The waveguide mode excitation layer is configured by laminating a first layer and a second layer in this order on the light transmissive substrate, and is a layer that can excite the waveguide mode.
The waveguide mode is excited by irradiating the sensor chip with light from the light transmissive substrate side (back side) under total reflection conditions.

--first layer--
The first layer is formed of one of a metal material, a semiconductor material and a first dielectric material.
The metal material is not particularly limited, and examples thereof include metal materials such as gold, silver, and copper.
The semiconductor material is not particularly limited, and includes known semiconductor materials such as Si, Ge, and SiGe, and compound semiconductor materials. Among them, Si and Ge, which easily obtain a high refractive index of 3.0 or more, are preferable. .
The first dielectric material is not particularly limited, and examples thereof include known light-transmitting dielectric materials such as TiO ₂ and Ta ₂ O ₅ . is easy to obtain and TiO ₂ with a low extinction coefficient (high light transmittance) is preferable.
Further, it is known that the optimum value of the thickness of the first layer is determined depending on the constituent material and the wavelength of the light to be irradiated, and that this value can be calculated from the calculation using the Fresnel equation. there is In general, when light in a wavelength band from near ultraviolet to near infrared is used, the first thickness is several nanometers to several hundred nanometers.
The method for forming the first layer is not particularly limited, and can be appropriately selected according to the material to be formed. Examples include known methods such as bonding, sputtering, and vapor deposition. can.

--Second layer--
The second layer is formed of a second dielectric material.
The second dielectric material is not particularly limited, and examples thereof include glass materials such as silica glass, oxides such as TiO ₂ , nitrides such as AlN, and fluorides such as MgF ₂ and CaF. Among them, silica glass having a low extinction coefficient is preferable. Examples of the silica glass include silica glass itself formed of silicon dioxide (SiO ₂ ) as a single component, and glass obtained by adding an additive to silica glass such as boron silica glass, fluorine-added silica glass, and germanium-added silica glass. including.
As for the thickness of the second layer, the optimum value is determined by the constituent material and the wavelength of the light to be irradiated, as in the case of the first layer. known to be possible. Generally, the thickness of the second layer is several tens of nm to several μm.
The method for forming the second layer is not particularly limited and can be appropriately selected according to the forming material. Examples thereof include known methods such as sputtering and spin coating. Further, when the first layer is made of a semiconductor, the second layer can be formed by thermally oxidizing the first layer.

Here, when the light-transmissive substrate is a plate having parallel front and back surfaces, the light irradiated from the back surface side is not totally reflected if liquid exists on the surface. Therefore, in such a case, by forming a diffraction grating on the back surface portion of the light-transmitting substrate, when the diffraction grating is irradiated with light at a specific angle, the light is diffracted by the diffraction grating to form the sensor chip. and the sensor chip may be configured such that light introduced into the sensor chip illuminates the surface under conditions of total internal reflection to excite guided modes in said guided mode excitation layer. . Alternatively, the front surface and the rear surface of the light transmissive substrate may be formed so as not to be parallel to each other. Alternatively, the back surface of the detection plate may be irradiated with the light emitted from the light source through a known prism. The prism can be used by being optically attached to the back surface of the sensor chip with a refractive index adjustment oil, an optical adhesive, or the like. When the same forming material as that of the light-transmitting substrate is selected as the forming material of the prisms, the light-transmitting substrate and the prisms can be integrally molded.

<Liquid holding part>
A liquid sample (liquid mixture) is introduced to the surface of the sensor chip on the waveguide mode excitation layer side, that is, the surface of the sensor chip. As a method for holding the liquid sample on the surface of the sensor chip, for example, the liquid sample is dropped onto the surface of the sensor chip and then covered with a cover glass or the like.
Further, a liquid sample reservoir may be formed on the surface of the sensor chip in order to securely hold the liquid sample. The liquid sample tank is not particularly limited. It is preferable that a side wall portion, which is a constituent portion of the liquid sample tank, is arranged.
The material for forming the side wall portion is not particularly limited, and known glass materials, resin materials, and the like can be mentioned. can.

<Light irradiation part>
The light irradiation section is a section that can irradiate the sensor chip with light from the light transmissive substrate side through the optical prism under a condition of total reflection.
The light source of the light irradiation section is not particularly limited and can be appropriately selected according to the purpose, and examples thereof include known lamps, LEDs, lasers, and the like.
A waveguide mode is formed in the first layer and the second layer of the sensor chip when the sensor chip is irradiated with light through the optical prism under the condition of total reflection.
Therefore, in this case, the role required of the light irradiation unit is only to irradiate the sensor chip with light under the condition of total reflection via the optical prism. there is no limit to

When using a radiation light source such as a lamp or LED, it is preferable to keep the incident angle constant. For this reason, when a radiation light source is used, it is preferable to use a guide part such as a collimating lens that regulates the irradiation direction of the irradiation light to a specific direction.

<Photodetector>
The light detection unit detects reflected light of light irradiated from the rear surface side of the sensor chip under a total reflection condition and the target substance emitted based on irradiation of the sensor chip with light under the total reflection condition from the rear surface side. It is a portion that detects either fluorescence or scattered light that accompanies the presence of .
The photodetector is not particularly limited, and photodetectors such as photodiodes, photomultiplier tubes, CCDs, and CMOS image sensors can be used. Moreover, when measuring the wavelength dependence of the detected light intensity by observing the reflected light spectrum, it is preferable to use a spectroscope in the light detection section.

Also, the configuration of the photodetector is set according to the sensing mode of the waveguide mode sensor.
That is, as the sensing mode by the waveguide mode sensor, there is a type that detects the reflected light of the light irradiated from the back side of the sensor chip under the total reflection condition, and a type that detects the light reflected from the back side of the sensor chip under the total reflection condition. There are two main types of fluorescence or scattered light associated with the presence of the target substance emitted based on irradiating .
In the type that detects the reflected light, the photodetector is arranged on the path of the reflected light, and in the type that detects the fluorescence or the scattered light, the surface of the sensor chip from which the fluorescence or the scattered light is emitted. By placing the photodetector on top, these two types of guided mode sensors are constructed.

(Target substance detection method)
The target substance detection method of the present invention will be described below, along with a description of the waveguide mode sensor suitably used for the target substance detection method.

The target substance detection method includes a biotin immobilization step, a mixture preparation step, a mixture introduction step, and a target substance detection step.

-Biotin immobilization step-
The biotin immobilization step is a step of immobilizing biotin on the surface of the sensor chip of the waveguide mode sensor.
The biotin has a relatively low molecular weight and high stability, and the amount of the biotin immobilized on the surface of the sensor chip is easy to control. can be provided.
A method for immobilizing the biotin on the surface of the sensor chip is not particularly limited, and can be appropriately selected from known immobilization methods.
An example of the known immobilization method is as follows.

First, the sensor chip is ultrasonically cleaned with acetone and then ultrasonically cleaned with ethanol to clean the surface of the sensor chip.
Next, the sensor chip was immersed in a 0.1% by volume ethanol solution of 3-Aminopropyltriethoxysilane (APTES, manufactured by Shin-Etsu Chemical Co., Ltd., LS-3150) at room temperature for 24 hours to fix 3-Aminopropyltriethoxysilane on the surface of the sensor chip. Then, the surface of the sensor chip is aminated to modify the surface.
Then, the sensor chip removed from the solution is rinsed with acetone, ultrasonically cleaned in ethanol for 3 minutes, ultrasonically cleaned in 1 mM NaOH solution for 3 minutes, and cleaned in 1 mM HCl solution for 3 minutes. , ultrasonic cleaning in ultrapure water for 3 minutes, and drying by nitrogen blow are performed in this order.
Next, Biotin-(AC ₅ ) ₂ Sulfo-Osu (manufactured by Dojindo Laboratories, C ₂₆ H ₄₀ N ₅ NaO ₁₀ S ₂ =669.75), a compound having a biotin moiety, was added to phosphate buffered saline (PBS , Wako Pure Chemical Industries, Ltd. 10x PBS (-) diluted 10 times), and the sensor chip is immersed in a solution containing 350 µg/mL at room temperature for 24 hours.
Next, the sensor chip removed from the solution is rinsed with ultrapure water containing 0.05% by mass of Tween 20 (Tokyo Chemical Industry Co., Ltd., product code T0543), rinsed three times with ultrapure water, and dried. Do it in this order.
By the above procedure, the biotin is immobilized on the surface of the sensor chip.

The known immobilization method generally includes a first surface modification step of immobilizing a compound having an amino group on the sensor chip surface to aminate the sensor chip surface; It is carried out in a multi-step with a second surface modification step of binding with an amino group and biotinylating the sensor chip surface.
However, the implementation of each of the surface modification steps requires relatively complicated manipulations, the reproducibility of the biotin immobilization is low, and optimization is required.
In addition, when mass-producing the sensor chips with the biotin immobilized on the surface, the number of steps increases and the number of control items for suppressing variations between products increases, which is disadvantageous in terms of stable product supply and yield improvement. becomes.

Therefore, a one-step method of biotinylating the sensor chip surface in one step is preferred.
In the one-step method, the biotin immobilization step is carried out as a step of drying the sensor chip after contacting the surface of the sensor chip with a solution of a biotinylated silane coupling agent in which biotin is added to a silane compound. be.
That is, by using the biotinylated silane coupling agent having both a silane moiety and a biotin moiety that bind to the surface of the sensor chip, immobilization of the biotin on the surface of the sensor chip, which has been carried out in multiple steps until now, has been achieved. The process of doing is performed in one step.

As the biotinylated silane coupling agent, any agent having both the silane moiety and the biotin moiety can be used without particular limitation.
For example, a biotinylated silane coupling agent represented by the following general formula (A) can be used.
However, in the general formula (A), X represents a halogen atom such as F, Cl, Br, Y represents an alkoxy group such as an ethoxy group, R represents an alkyl group, an ethylene glycol chain, an aralkyl chain, It represents a divalent chain group such as an ether chain or thioether chain, and n represents an integer of 0-3.

Among them, a compound newly synthesized by the present inventors and represented by the following general formula (1) is preferable.
However, in the general formula (1), R ¹ represents an alkylene group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 2 to 15 carbon atoms.

The synthesis method of 11-biotinylated aminoundecane (triethoxysilylpropyl) thioether (hereinafter referred to as 11-biotinylated silane coupling agent) will be described below as a representative example of the biotinylated silane coupling agent represented by the general formula (1). However, by selecting the carbon chain length of the starting material, a biotinylated silane coupling agent of any carbon chain length corresponding to R ¹ and R ² in the general formula (1) can be added to the synthesis method. can be synthesized according to

<Synthesis of 11-biotinylated silane coupling agent>
(1) First, 11-undecene azide is synthesized according to the following scheme (1).

Specifically, first, 2.33 g (10 mmol) of 11-bromoundecene and 80 mL of DMF are placed in a three-necked flask (100 mL) and stirred at room temperature. Then add 1.30 g (20 mmol) of sodium azide and stir at 80° C. for 12 hours. After cooling, the reaction solution is poured into 100 mL of 5% by mass hydrochloric acid and extracted with 100 mL of chloroform. Then, chloroform and DMF are distilled off and dried under reduced pressure to synthesize crude 11-undecene azide.
The crude 11-undecene azide actually synthesized by this method was obtained as follows.
・Colorless liquid ・Crude yield 100%
- _{C11H21N3 ,} M = _195.31

(2) Next, 11-aminoundecene is synthesized according to the following scheme (2).

Specifically, first, 1.95 g (10 mmol) of crude 11-undecene azide, 4 mL of water, and 100 mL of THF are placed in a three-necked flask (300 mL) and stirred at 0°C. A solution of 3.93 g (15 mmol) of triphenylphosphine in 25 mL of THF is then added dropwise and stirred at room temperature for 12 hours. Then, THF is distilled off, and the 11-aminoundecene is purified using a silica gel column (concentration gradient; chloroform:methanol=100:2→100:100).
The 11-aminoundecene actually synthesized by this method was obtained as follows.
・Pale yellow liquid ・Yield 83%
- _C11H23N , M = _169.31

(3) Next, biotinamide undecene is synthesized according to the following scheme (3).

Specifically, first, 488 mg (2 mmol) of biotin, 339 mg (2 mmol) of 11-aminoundecene, 324 mg (2.4 mmol) of hydroxybenzotriazole (HOBt), and diisopropylethylamine (DIEA) were placed in a three-necked flask (100 mL) under a nitrogen atmosphere. ) 310 mg (2.4 mmol) and 30 mL of dehydrated DMF are added and ice-cooled. Next, 30 mL of a dehydrated DMF solution containing 934 mg (2.2 mmol) of benzotriazolyltetramethyluronium hexafluorophosphate (HBTU) is added dropwise, and the mixture is stirred at room temperature for 12 hours under a nitrogen atmosphere. DMF is distilled off, the resulting residue is poured into 100 mL of 5% by mass hydrochloric acid, extracted with 100 mL of chloroform, and the chloroform layer is washed three times with 100 mL of 5% by mass hydrochloric acid. Chloroform is distilled off, and the biotinamide undecene is purified using a silica gel column (concentration gradient; chloroform:methanol=100:2→100:5).
The biotinamide undecene actually synthesized by this method was obtained as follows.
・Colorless solid (gel-like)
・Yield 70%
- _{C21H37N3O2S ,} M _{= 395.60}

(4) Next, the 11-biotinylated silane coupling agent is synthesized according to the following scheme (4).

Specifically, 199 mg (0.5 mmol) of the biotinamide undecene, 5.1 mg (0.02 mmol) of 2,2'-dimethoxy-2-phenylacetophenone (DMPA), mercaptopropyltriethoxy were added to an eggplant flask (50 mL). 238 mg (1 mmol) of silane and 30 mL of dehydrated dichloromethane are added, and the dichloromethane is distilled off while stirring quickly. Then, the 11-biotinylated silane coupling agent is purified using a silica gel column (concentration gradient; chloroform:ethanol=100:2→100:10).
The 11-biotinylated silane coupling agent actually synthesized by this technique was obtained as follows.
・Colorless solid ・Yield 66%
- _{C30H59N3O5S2Si , M = 634.02}

Next, a procedure for biotinylating the surface of the sensor chip with the 11-biotinylated silane coupling agent will be described.
First, the sensor chip is ultrasonically cleaned with acetone and then ultrasonically cleaned with ethanol to clean the surface of the sensor chip.
Next, the sensor chip is immersed in a toluene solution containing 1 mM of the 11-biotinylated silane coupling agent and left at 20° C. for two weeks. This standing period can be shortened to 24 hours by setting the temperature at the time of immersion to 40°C.
After immersion, the sensor chip is taken out of the solution, rinsed with toluene, rinsed with acetone, and dried by blowing nitrogen.
Through the above process, the biotin can be immobilized on the surface of the sensor chip. In the method using the 11-biotinylated silane coupling agent, biotinylation can be performed only by immersing the sensor chip in the toluene solution for a predetermined time, then rinsing and drying (one step). can overcome the disadvantages in the immobilization method (multi-step) of
That is, in the one-step method, the process is simpler than the known immobilization method (multi-step), the reproducibility is high, and variations in products can be suppressed during mass production.

From this point of view, it is preferable to use the waveguide mode sensor provided with the sensor chip surface-modified with the biotinylated silane coupling agent. , is particularly preferably a compound represented by the general formula (1).

<Mixed liquid preparation process>
In the mixed solution preparation step, the subject whose presence of the target substance to be detected is verified is bound to at least a biotin conjugate selected from streptavidin and avidin and the biotin conjugate, and the It is a step of preparing a mixed solution in which a reagent containing a first target substance-capturing substance capable of binding and capturing a target substance is mixed.

The first target substance-capturing substance is not particularly limited as long as it is a substance that specifically captures the target substance, and can be appropriately selected according to the setting and purpose of the target substance. Examples include antibodies and aptamers against
The method for adding the biotin conjugate to the first target substance-capturing substance is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a method using a commercially available streptavidin labeling kit. be done.

The reagent may further include a labeling substance that can be sensed by the waveguide mode sensor, and a second target substance-capturing substance that binds to the labeling substance and binds to the target substance to enable capture. .
The labeling substance is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include dyes, colored beads, metal nanoparticles and the like. Among these, dyes and nanoparticles that absorb light in the visible light region, dyes and fine particles that emit fluorescence, and fine particles that emit scattered light are preferred, and gold nanoparticles are particularly preferred. Fine particles refer to particles having a maximum diameter of 1 nm to 1 μm.
The shape of the gold nanoparticles is not particularly limited, and may be spherical, ellipsoidal, plate-like, or the like.
The size of the gold nanoparticles is preferably 6.5×10 ⁻²⁰ cm ³ to 1.1×10 ⁻¹⁶ cm ³ in terms of volume since there is no limitation on the shape. This size corresponds to a diameter of 5 nm to 60 nm when the gold nanoparticles are spherical in shape. Preferred sizes for the spherical shape will be described below.
That is, when the diameter of the spherical gold nanoparticles is less than 5 nm, the signal tends to be weak and difficult to detect, and it tends to be difficult to obtain particles with a uniform particle size.
In addition, when the diameter of the spherical gold nanoparticles exceeds 60 nm, sedimentation due to gravity may overcome Brownian motion and sink to the surface of the sensor chip due to its own weight. Particles that have sunk due to their own weight are detected as signals, causing false detection. From this point of view, it is more preferable that the spherical gold nanoparticles have a diameter of 40 nm or less. However, even when the diameter of the spherical gold nanoparticles exceeds 40 nm, if the diameter is 60 nm or less, the sedimentation velocity is slow, and thus the measurement can be completed within several minutes. The spherical gold nanoparticles with a diameter of 40 nm are converted to a volume of 3.4×10 ⁻¹⁷ cm ³ .

The labeling substance is not necessary when the target substance itself imparts a characteristic change of the reflected light or emits the fluorescence or the scattered light. Used for sensing in the waveguide mode sensor.
For example, a case where the target substance is a transparent substance such as protein will be described with reference to FIG.
FIG. 1 is a diagram showing an example of detection of a dip caused by a sudden decrease in reflected light intensity at a specific wavelength when light having broad wavelength characteristics is incident at a specific incident angle.
When the transparent substance is adsorbed on the surface of the sensor chip, the position of the dip moves in the lateral direction (long wavelength direction). On the other hand, when a substance (labeling substance) having light absorption in this wavelength band is adsorbed on the sensor chip surface, the depth of the dip bottom becomes deeper.
In this case, the dip changes in the following manner depending on whether the target substance is present or not.
That is, in the absence of the target substance, the first target capturing substance (such as an antibody) with the biotin conjugate (such as the streptavidin) is adsorbed to the surface of the sensor chip. If both the first target capture substance is transparent, the dip moves laterally.
On the other hand, when the target substance is present, the conjugate of the first target capturing substance with the biotin conjugate--the target substance--the labeling substance is adsorbed to the surface of the sensor chip. Since the body, the first target-capturing substance and the target substance are transparent, the dip moves laterally, but since the labeling substance uses a substance with light absorption, the depth of the bottom of the dip is greater. shift in any direction.
From the above, the presence or absence of the target substance cannot be determined by focusing on the lateral movement of the dip, but the presence of the target substance can be detected by monitoring the depth of the dip. The amount of the target substance can be measured from the amount of change in thickness.
Furthermore, the subject may contain impurities, which are generally transparent substances in many cases. Therefore, using the method of measuring the depth of the dip has the advantage of being less susceptible to the adsorption of impurities.

As the labeling substance, if the labeling substance itself has the property of being adsorbed to the target substance, it can be used as it is. However, when the labeling substance itself does not have the property of being adsorbed to the target substance, the second target substance-capturing substance that binds to the labeling substance and binds to the target substance to enable capture is used. Add and use.
The second target substance-capturing substance is not particularly limited as long as it binds to the labeling substance and specifically captures the target substance. Antibodies, aptamers, and the like against the target substance can be selected as appropriate.

By the way, the labeling substance and the second target substance-capturing substance with the labeling substance are not included in the mixed solution, and the biotin-biotin conjugate is added to the surface of the sensor chip by the mixed solution introducing step described later. In a state in which the first target substance-capturing substance-marking substance conjugate is formed, it is also possible to introduce an introduction liquid containing these onto the sensor chip surface.
However, in the prior art, the mixed solution and the introduction solution are handled separately, as in the case of separately handling the reagent in which the antibody is bound to streptavidin or avidin on the surface of the sensor chip, and the sample solution. If handled separately, it is necessary to mix the mixed solution and the introduced solution for binding the target substance and the second target substance-capturing substance on the surface of the sensor chip where the space is limited. Therefore, the work before detection processing is complicated.
Therefore, it is preferable that the labeling substance and the second target substance-capturing substance with the labeling substance are contained in the reagent and handled in one mixed solution.

<Mixed solution introduction step>
The mixed solution introducing step is a step of introducing the mixed solution onto the surface of the sensor chip on which the biotin is immobilized.
When the mixed solution is introduced onto the surface of the sensor chip, depending on the components contained in the mixed solution, the first target capturing substance with the biotin conjugate - the target substance, the biotin conjugate with The first target capturing substance-the target substance-the labeling substance conjugate and the first target capturing substance with the biotin conjugate-the target substance-the second target substance-capturing substance with the labeling substance is immobilized on the sensor chip surface via the biotin conjugate.
Therefore, the pretreatment before sensing by the waveguide mode sensor can be significantly simplified.

The method for introducing the mixed solution is not particularly limited, and may be covering and holding the mixed solution introduced onto the surface of the sensor chip with a cover glass, or forming the liquid sample tank on the sensor chip. In some cases, the liquid mixture may be introduced into the liquid sample tank.
After introducing the mixed solution onto the surface of the sensor chip, the mixed solution may be allowed to stand still for a period of time necessary for the binding of the biotin and the biotin conjugate. The liquid mixture introduced onto the surface of the sensor chip may be stirred by using a

<Target substance detection step>
The target substance detection step is any one of the mixed solution containing no target substance, the reagent not mixed with the specimen, and the mixed solution in which the content of the target substance is controlled. The sensing state of the waveguide mode sensor when the reference liquid is introduced onto the surface of the sensor chip on which the biotin is immobilized is defined as the reference state, and the waveguide when the mixed liquid is introduced in the mixed liquid introduction step. A step of detecting the target substance by comparing the sensing state of the mode sensor as the measurement state with the reference state and the measurement state.

By selecting the mixed solution that does not contain the target substance and the reagent that is not mixed with the specimen as the reference solution, the presence or absence of the target substance and the amount of the target substance can be detected. Selecting the mixed solution in which the content of the target substance is controlled as the solution is useful for quantifying the amount of the target substance present.
The reference solution is used for quantifying the presence or absence of the target substance and the amount of the target substance by sensing using the waveguide mode sensor, and additives that do not affect sensing are added. may

The target substance detection step can be performed according to the sensing mode of the waveguide mode sensor.
That is, according to the first method regarding the implementation method of sensing by the waveguide mode sensor, the sensing by the waveguide mode sensor detects the reflected light of the light irradiated from the rear surface side of the sensor chip under total reflection conditions. is implemented as
In this case, as described with reference to FIG. 1, from the viewpoint of suppressing the influence of impurity adsorption, the target substance detection step includes It is preferable to compare the depth of the dip bottom.
Further, according to the second method relating to the method of performing sensing by the waveguide mode sensor, the sensing by the waveguide mode sensor is based on irradiating the sensor chip with light from the rear surface side under total reflection conditions. detection is performed by detecting the fluorescence or the scattered light associated with the presence of the target substance emitted by

(First embodiment)
Next, a first embodiment of the invention will be described with reference to FIGS. 2 to 5. FIG. FIG. 2 is an explanatory diagram for explaining the first embodiment, FIG. 3 is an explanatory diagram showing an overview of the biotin immobilization step, and FIG. 4 is an explanation showing an overview of the mixture preparation step. FIG. 5 is an explanatory diagram showing an outline of the mixed liquid introducing step.

As shown in FIG. 2, the waveguide mode sensor 100 has a sensor chip 101, a light irradiation section 104, a light detection section 105, and an optical prism .
Further, in the waveguide mode sensor 100 , the liquid mixture 102 is held on the surface of the sensor chip 101 by covering the liquid mixture 102 introduced onto the surface of the sensor chip 101 with the cover glass 103 .
The waveguide mode sensor 100 is a type of waveguide mode sensor that detects the reflected light of the light irradiated from the back side of the sensor chip 101 (the side in contact with the optical prism 106) under total reflection conditions.

In the target substance detection method according to the first embodiment, as shown in FIG. 3, first, before the mixture 102 is introduced onto the surface of the sensor chip 101, biotin 3 is immobilized on the surface of the sensor chip 101. (Biotin immobilization step). In FIG. 3, the aminated surface modification layer 2 is once formed on the surface of the sensor chip 101 by the multi-step, and then the biotin 3 is formed on the aminated surface modification layer 2. Biotin 3 can also be directly immobilized on the surface of the sensor chip 101 by the one step using the ring agent.
Next, as shown in FIG. 4, the test subject for which the presence of a target substance 13 (for example, an antigen) to be detected is verified, and the biotin conjugate 11 selected from either the streptavidin or the avidin. , a first target substance capturing substance 12 (for example, an antibody) that is bound to the biotin conjugate 11 and is capable of being captured by binding to the target substance 13, and a labeling substance 15 that can be sensed by the waveguide mode sensor 100. (e.g., gold nanoparticles) and a second target substance-capturing substance 14 (e.g., antibody) that binds to the labeling substance 15 and binds to the target substance 13 so that it can be captured is mixed. prepare the mixed liquid 102 (mixed liquid preparation step)
Next, as shown in FIG. 5, mixed liquid 102 is introduced onto the surface of sensor chip 101 .
When mixed solution 102 is introduced, biotin 3 immobilized on the surface of sensor chip 101, biotin-conjugate 11, first target substance-capturing substance 12, target substance 13, and second target substance-capturing substance 14 are combined. and a labeling substance in this order are bound via the biotin conjugate 11, and the pretreatment for detecting the presence of the target substance 13 is completed.
That is, in the target substance detection method according to the first embodiment, the pretreatment for detecting the presence of the target substance 13 can be completed simply by introducing the mixed liquid 102 onto the surface of the sensor chip 101. Detection operation of the target substance 13 using the wave mode sensor 100 can be significantly simplified.

(Second embodiment)
Next, a second embodiment of the invention will be described with reference to FIG. Note that FIG. 6 is an explanatory diagram for explaining the second embodiment.
As shown in FIG. 6, waveguide mode sensor 200 has sensor chip 201 , light irradiation section 204 , light detection section 205 and optical prism 206 .
In the waveguide mode sensor 200 , the liquid mixture 202 is held on the surface of the sensor chip 201 by covering the liquid mixture 202 introduced onto the surface of the sensor chip 201 with the cover glass 203 .
This waveguide mode sensor 200 detects the fluorescence or the scattering associated with the presence of the target substance emitted based on irradiation of the sensor chip 201 with light from the rear surface side (the side in contact with the optical prism 206) under total internal reflection conditions. It becomes a type of waveguide mode sensor that detects light.
That is, unlike the waveguide mode sensor 100 in which the light detection section 105 is arranged on the path of the reflected light for detecting the reflected light, the waveguide mode sensor 200 detects the fluorescence or the scattered light ("L A photodetector 205 is arranged on the sensor chip 201 for detecting the light indicated by .
Since other items are the same as those of the waveguide mode sensor 100, description thereof is omitted.

( Reference example 1)
As Reference Example 1, the target substance detection method of the present invention was carried out as follows.
As a waveguide mode sensor, a waveguide mode sensor (Eva-M01) manufactured by C&I was used. As a sensor chip, a waveguide mode sensor chip (M01-35-40-325-01) manufactured by C&I was used.
First, the sensor chip was ultrasonically cleaned with acetone and then ultrasonically cleaned with ethanol to clean the surface of the sensor chip.
Next, the sensor chip was immersed in a 0.1% by volume ethanol solution of 3-Aminopropyltriethoxysilane (APTES, manufactured by Shin-Etsu Chemical Co., Ltd., LS-3150) at room temperature for 24 hours to fix 3-Aminopropyltriethoxysilane on the surface of the sensor chip. Surface modification was carried out to aminate the surface of the sensor chip.
Then, the sensor chip was removed from the solution, rinsed with acetone, ultrasonically cleaned in ethanol for 3 minutes, ultrasonically cleaned in 1 mM NaOH solution for 3 minutes, and cleaned in 1 mM HCl solution for 3 minutes. Ultrasonic cleaning, ultrasonic cleaning in ultrapure water for 3 minutes, and drying by nitrogen blowing were performed in this order.
Next, the sensor chip was placed in a 350 μg/mL PBS solution of Biotin-(AC ₅ ) ₂ Sulfo-Osu (manufactured by Dojindo Laboratories, C ₂₆ H ₄₀ N ₅ NaO ₁₀ S ₂ =669.75), a compound having a biotin site. It was immersed at room temperature for 24 hours.
Next, the sensor chip removed from the solution was rinsed with ultrapure water containing 0.05% by mass of Tween 20, rinsed three times with ultrapure water, and dried in this order.
By the above procedure, biotin was immobilized on the surface of the sensor chip.

A CRP antigen (C-reactive protein, manufactured by Fitzgerald) was used as a target substance to be detected.

Anti-h CRP clone 6405 SPTN-5 (code: 100358, manufactured by Medix, hereinafter referred to as "6405 antibody") was used as the first target substance-capturing substance. A first target substance-capturing substance with streptavidin (hereinafter referred to as "primary antibody") was prepared as follows.
A streptavidin labeling kit (FastLink Streptavidin Labeling Kit, Abnova) was used as a kit for attaching streptavidin to the 6405 antibody. The procedure attached to the 6405 antibody with streptavidin was carried out according to the protocol attached to the same kit. Specifically, it is as follows.
First, 10 μL of Modifier Reagent is added to 100 μL of a solution of 6405 antibody diluted to 1 mg/mL with PBS and mixed. The entire amount of this mixture is added to a glass vial containing Fast Link Mix (NHS-activated streptavidin lyophilized powder), mixed well, and allowed to react overnight at 4°C.
Next, 1/10 volume of Quencher Reagent per reaction solution is added and thoroughly mixed, and the mixture is allowed to stand at room temperature for 30 minutes to terminate the reaction.

Gold nanocolloid was selected as the labeling substance.
Anti-h CRP clone 6404 SP-6 (code: 100061, manufactured by Medix, hereinafter referred to as "6404 antibody") was bound to the labeled substance as a second target substance-capturing substance. The gold nanocolloid-attached 6404 antibody prepared here is called a secondary antibody.
A gold nanocolloid labeling kit (20 nm NHS-Activated Gold Nanoparticle Conjugation Kit, manufactured by CTD) was used to prepare the secondary antibody. The secondary antibody was prepared according to the protocol attached to the kit. Specifically, it is as follows.
First, the 6404 antibody solution is dialyzed and replaced with Buffer A (20 mM NaHCO ₃ , 0.1 M NaCl).
Next, 90 μL of 108 μL obtained by thoroughly mixing 48 μL of the antibody solution after dialysis and 60 μL of Reaction Buffer is added to a vial of lyophilized NHS-activated gold nanocolloids and thoroughly mixed. React overnight at 4°C.
Then, after adding 10 μL of Quencher solution and sufficiently mixing, centrifugation is performed under conditions of 7,500 rpm, 20° C. and 60 minutes, and the supernatant is removed.
Next, after adding Buffer B (20 mM Tris, 150 mM NaCl, 1% by mass BSA, 0.05% by mass PEG20000, pH 8.2) to the residue, centrifugation was performed at 7,500 rpm, 20°C for 60 minutes. and remove the supernatant.
Subsequently, the residue is subjected to the same supernatant removal operation twice more to remove the labeling substance and unreacted 6404 antibody.
Then redissolve in Buffer B.
A secondary antibody was prepared by the above procedure.

The prepared primary antibody and secondary antibody were diluted with 0.01% by mass Tween PBS, and adjusted to a final concentration of 2 nM and 200 pM when mixed, respectively.
In addition, the CRP antigen was diluted with 0.01% by mass Tween PBS, and adjusted to four final concentrations of 10 pM, 50 pM, 100 pM and 200 pM when mixed.
As a negative control, 0.01 mass % Tween PBS containing no CRP antigen was used.
As a positive control, a solution containing 200 pM of a conjugate (SA-Au) obtained by directly adsorbing streptavidin to gold nanocolloid was used.
Sensing by the waveguide mode sensor (Eva-M01) is performed by mixing the diluted solutions containing the primary antibody, secondary antibody and CRP antigen in a tube and allowing the mixture to stand for 30 minutes. 40 μL was dropped on the sensor chip of the waveguide mode sensor.
In addition, for sensing with a waveguide mode sensor (Eva-M01), we used the built-in function of the same sensor to trace the change in reflectance at the dip position in the reflection spectrum caused by waveguide mode excitation. .
When the conjugate of CRP antigen, primary antibody and secondary antibody binds to biotin immobilized on the sensor chip surface by streptavidin, the gold nanocolloid effect causes a dip position in the reflection spectrum caused by waveguide mode excitation. reflectance decreases. That is, the position of the bottom of the dip in the reflected light spectrum is deeper than when the conjugate of the CRP antigen, the primary antibody, and the secondary antibody is not bound to the biotin immobilized on the surface of the sensor chip by streptavidin. becomes.

FIG. 7 is a diagram showing the measurement results at the dip position of the reflected light spectrum for negative control, positive control, and CRP antigen 100 pM and 200 pM samples.
As shown in FIG. 7, it can be seen that the concentration-dependent CRP antigen can be detected.

FIG. 8 shows the measurement results at the dip positions of the reflected light spectrum for the negative control and the CRP antigen 10 pM and 50 pM samples.
As shown in FIG. 8, even when the concentration of the CRP antigen was as low as 10 pM, a clear difference from the negative control was obtained, indicating successful detection. In addition, when comparing the case of CRP antigen 10 pM and the case of CRP antigen 50 pM, it can be seen that the signal intensity increases according to the concentration.

According to the method of Reference Example 1 shown above, the detection and quantitative evaluation of the target substance can be performed only by mixing the analyte with the reagent containing the primary antibody and the secondary antibody and introducing it onto the surface of the sensor chip. confirmed to be possible.

(Example 1 )
In Example 1 , instead of the method of Reference Example 1, the method of immobilizing biotin on the surface of the sensor chip was as follows.
First, the sensor chip was ultrasonically cleaned with acetone and then ultrasonically cleaned with ethanol to clean the surface of the sensor chip.
Next, the sensor chip was immersed in a toluene solution containing 1 mM of the aforementioned 11-biotinylated silane coupling agent and left at 20° C. for two weeks.
Next, the sensor chip was taken out of the solution and subjected to rinsing with toluene, rinsing with acetone, and drying by blowing nitrogen in this order.
Biotin was immobilized on the surface of the sensor chip by the above procedure. The procedure in Example 1 does not require surface modification such as amination of the surface of the sensor chip, and the operation is simpler than the procedure in Reference Example 1 in which this treatment is performed.

As the CRP antigen, primary antibody and secondary antibody, those prepared in Reference Example 1 were used.
Also, the same sensor and sensor chip as those used in Reference Example 1 were used, and similar sensing was performed.
Also, the primary antibody and secondary antibody were diluted with 0.01% by mass Tween PBS and adjusted to final concentrations of 2 nM and 600 pM when mixed, respectively.
Also, the CRP antigen was diluted with 0.01% by mass Tween PBS and adjusted to a final concentration of 300 pM when mixed.
As a negative control, 0.01% by mass Tween PBS containing no CRP antigen was used.
As a positive control, a solution containing 600 pM of a conjugate (SA-Au) obtained by directly adsorbing streptavidin to gold nanocolloid was used.

FIG. 9 is a diagram showing the measurement results at the dip position of the reflected light spectra for the negative control, positive control, and 300 pM CRP antigen sample.
As shown in FIG. 9, it can be seen that 300 pM CRP antigen was detected.

Reference Signs List 101, 201 sensor chip 2 aminated surface modification layer 3 biotin 102, 202 mixed solution 11 biotin conjugate 12 first target substance capturing substance 13 target substance 14 second target substance capturing substance 15 labeling substance 100, 200 waveguide mode Sensor 103, 203 Cover glass 104, 204 Light irradiation unit 105, 205 Light detection unit 106, 206 Optical prism

Claims (8)

a biotin immobilization step of immobilizing biotin on the surface of the sensor chip of the waveguide mode sensor;
A subject whose presence of a target substance to be detected is verified, a biotin conjugate selected from at least one of streptavidin and avidin, and a biotin conjugate bound to the biotin conjugate and capable of binding and capturing the target substance. a mixed solution preparation step of preparing a mixed solution in which the reagent containing the first target substance-capturing substance is mixed;
a mixed solution introducing step of introducing the mixed solution onto the surface of the sensor chip on which the biotin is immobilized;
The biotin immobilizes the reference solution, which is any one of the mixed solution without the target substance, the reagent not mixed with the specimen, and the mixed solution in which the content of the target substance is controlled. The sensing state of the waveguide mode sensor when the mixed liquid is introduced onto the surface of the sensor chip thus formed is defined as a reference state, and the sensing state of the waveguide mode sensor when the mixed liquid is introduced in the mixed liquid introduction step is defined as a reference state. a target substance detection step of detecting the target substance by comparing the reference state and the measurement state as the measurement state;
including
The biotin immobilization step is a step of drying the sensor chip after contacting the surface of the sensor chip with a solution of a biotinylated silane coupling agent in which biotin is added to a silane compound ,
A target substance detection method , wherein the biotinylated silane coupling agent is a compound represented by the following general formula (1) .
However, in the general formula (1), R ¹ represents an alkylene group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 2 to 15 carbon atoms.

2. The reagent according to claim 1, further comprising a labeling substance that can be sensed by the waveguide mode sensor, and a second target substance-capturing substance that binds to the labeling substance and binds to the target substance to enable capture. target substance detection method. 3. The target substance detection method according to claim 2, wherein the first target substance-capturing substance and the second target substance-capturing substance are either an antibody or an aptamer against the target substance. 4. The target substance according to any one of claims 2 and 3, wherein the labeling substance is any one of dyes and nanoparticles that absorb light in the visible light region, dyes and fine particles that emit fluorescence, and fine particles that emit scattered light. Detection method. 5. The target substance detection method according to any one of claims 1 to 4, wherein sensing by the waveguide mode sensor is performed by detecting reflected light of light irradiated from the rear surface side of the sensor chip under total reflection conditions. 6. The target substance detection method according to claim 5, wherein the target substance detection step is performed by comparing the depth of the dip bottom in the reflected light spectrum of the reflected light detected in the reference state and the measurement state. . From claim 1, wherein the sensing by the waveguide mode sensor is performed by detecting fluorescence or scattered light accompanying the presence of the target substance, which is emitted based on irradiating the sensor chip with light from the rear surface side under conditions of total internal reflection. 5. The target substance detection method according to any one of 4. A sensor chip whose surface is modified with a biotinylated silane coupling agent in which biotin is added to a silane compound is arranged, and the biotinylated silane coupling agent is a compound represented by the following general formula (1). Guided mode sensor.
However, in the general formula (1), R ¹ represents an alkylene group having 2 to 10 carbon atoms, and R ² represents an alkylene group having 2 to 15 carbon atoms.

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