JP2013011465A - Manufacturing method of modified group introduced substrate, manufacturing method of ligand immobilization substrate, modified group introduced substrate and ligand immobilization substrate, and intermolecular interaction detecting method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a substrate facilitating a ligand immobilization operation to a substrate and easily assuring reproducibility of a ligand immobilization ratio, and to provide the substrate and an intermolecular interaction detecting method.SOLUTION: The manufacturing method of ligand immobilization substrates includes: a step (a) for preparing a substrate having a surface comprising SiN (silicon nitride); a step (b) for introducing at least one modified group selected from groups of an epoxy group, an azido group, an isocyanate group, and an isothiocyanate group into the substrate surface comprising SiN (silicon nitride); and, effectively, a step (c) for immobilizing ligands including at least one reactive group selected from groups comprising a thiol group, an amino group, and a hydroxyl group on the surface of the substrate via the reaction between the reactive group and the modified group.

Description

  The present invention relates to a method for producing a modified group-introducing substrate and a ligand-immobilized substrate suitable as a sensor chip for reflection interference spectroscopy (RIfS), a modified group-introducing substrate and a ligand-immobilized substrate obtained by the production method. And an intermolecular interaction detection method using the substrate.

  In recent years, research and development of a method for directly detecting a bond (intermolecular interaction) between a biomolecule and an organic polymer without using a label has been advanced. For example, RIfS using interference color change of an optical thin film has been proposed and put into practical use. The basic principle of the RIfS method is described in Patent Document 1 and the like. In addition, methods such as a surface plasmon resonance (SPR) method and a quartz crystal microbalance (QCM) method are known.

  In these intermolecular interaction detection devices, a substrate-like measurement member called a sensor chip, in which a capture substance (ligand) corresponding to a substance to be measured (analyte) is immobilized on the surface layer, is used. In general, antigen-antibody reaction, DNA hybridization, sugar / lectin interaction, and the like have been used as the intermolecular interaction between the substance to be measured and the capture substance.

For example, a protein (antibody) that can specifically bind to a surface of a sensor chip that uses a certain protein (antigen) as a substance to be measured is immobilized as a capture substance.
As a method for immobilizing a ligand such as a protein on a substrate, for example, the carboxyl group on the substrate surface is converted to N-hydroxy by using water-soluble carbodiimide (1-ethyl-3,4-dimethylaminopropylcarbodiimide [EDC]). Activated as an ester of succinimide [NHS] and reacted with the amino group of the ligand (Patent Documents 1 and 2) or the amino group of the substrate surface and the amino group of the ligand using glutaraldehyde which is a bifunctional reagent A method of cross-linking (Patent Document 3) is known.

  However, since EDC and glutaraldehyde used in these methods are susceptible to hydrolysis and oxidation and become unstable, it may be difficult to ensure the reproducibility of the immobilization rate. There is a problem that a difference occurs in the measured value. In addition, since the immobilization method is easily affected by the environment, there is a problem that it is not suitable for industrialization such as mass production of sensor chips.

JP 2004-346209 A (Patent No. 4009721) Special table 2007-514174 gazette Japanese Patent No. 35007048

  An object of the present invention is to provide a method for producing a substrate, in which the ligand immobilization operation to the substrate is simple and the reproducibility of the ligand immobilization rate is easily ensured, and the substrate and the intermolecular interaction detection method. To do.

  The present inventors have found that when an epoxy group is introduced on the substrate surface, ligands such as proteins can be immobilized on the substrate in one step without using reagents such as activated ester reagents (EDC, NHC) and glutaraldehyde, The present invention has been completed.

That is, the present invention includes the following matters.
[1] Step (a): preparing a substrate having a surface made of SiN [silicon nitride], and step (b): epoxy group, azide group, isocyanate group and isothiocyanate on the surface made of SiN of the substrate. And a step of introducing at least one modification group selected from the group consisting of groups.

[2] The production method according to [1], wherein the step (b) is performed using a silane coupling agent having the modifying group.
[3] The manufacturing method according to [1] or [2], wherein the substrate is a sensor chip for reflection interference spectroscopy (RIfS).

  [4] Step (c): A ligand having at least one reactive group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group, and the reactive group, according to any one of claims 1 to 3 A method for producing a ligand-immobilized substrate, comprising a step of immobilizing on a surface of the modifying group-introducing substrate through a reaction with a modifying group possessed by the modifying group-introducing substrate obtained by the production method.

[5] The production method according to [4], wherein the ligand is at least one selected from the group consisting of proteins, polypeptides, nucleic acids, and sugars.
[6] A modifying group-introducing substrate produced by the production method according to any one of [1] to [3].

[7] A ligand-immobilized substrate produced by the production method according to [4] or [5].
[8] A method for detecting an intermolecular interaction, comprising using the ligand-immobilized substrate according to [7].

According to the present invention, the modifying group introduced on the substrate surface reacts directly with the ligand, and the ligand can be immobilized on the substrate. Therefore, it is not necessary to use an unstable reagent.
Since the ligand can be immobilized on the substrate simply by immersing the substrate into which the modifying group is introduced in the ligand solution, there is no room for a difference in the immobilization rate by the operator, and stable operation is possible.

FIG. 1 is a view schematically showing a step (b) according to a method of manufacturing a substrate of this embodiment, particularly a sensor chip whose surface is made of SiN. FIG. 2 (a) shows a general chemical reaction formula between an amino group and an epoxy group, and FIG. 2 (b) shows an epoxy group introduced on the substrate surface used in this embodiment and an amino group possessed by a ligand (antibody). Shows a schematic view of the reaction of the ligand immobilized on the substrate. FIG. 3 shows a graph (a) indicating the timing of injecting the reagent into the RIfS type intermolecular interaction measuring apparatus in Example 1, and a graph (b) of the obtained results. FIG. 4 is a schematic diagram of an embodiment of an RIFS-based intermolecular interaction detection system.

Next, embodiments of the substrate manufacturing method, the substrate obtained by the manufacturing method, and the intermolecular interaction detection method using the substrate will be described in detail.
In the present specification, the “molecular interaction detection method” means that when some kind of interaction acts between a molecule provided on the surface of an analysis member (sensor chip) and a molecule to be analyzed. It is a method to detect the signal that appears. Such intermolecular interaction detection methods typically include RIfS, SPR, and QCM, but are not limited thereto. Further, the “intermolecular interaction detection device” is a device for carrying out the intermolecular interaction detection method described above.

<Manufacturing method of substrate and substrate thereof>
(Production method)
There are two types of substrate manufacturing methods in this embodiment.

The first method for producing a substrate is a method for producing a modifying group-introduced substrate, and includes the following steps (a) and (b).
Step (a): A step of preparing a substrate having a surface made of SiN [silicon nitride].

  Step (b): A step of introducing at least one modifying group selected from the group consisting of an epoxy group, an azide group, an isocyanate group, and an isothiocyanate group into the SiN surface of the substrate.

  In the step (a), typically, a SiN layer is formed on a Si substrate such as a silicon wafer by vapor deposition or the like to produce a substrate having a surface made of SiN. The substrate subjected to the step (b) is usually a so-called unmodified substrate in which the surface made of SiN has not been modified yet. For example, it is an unmodified sensor chip for RIfS that includes only a Si substrate and a SiN film formed on the Si substrate.

  The method for producing the second substrate is a method for producing a ligand-immobilized substrate, and further includes the following step (c) in addition to the method for producing the first substrate. Various conditions such as the concentration, pH, reaction temperature, and immersion time of the aqueous silane coupling agent solution can be adjusted to an appropriate range by those skilled in the art.

  Step (c): A ligand having at least one reactive group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group is reacted with the reactive group and the modifying group possessed by the modifying group-introducing substrate. And immobilizing on the surface of the modifying group-introducing substrate.

  As such a substrate, a sensor chip for reflection interference spectroscopy [RIfS] is suitable. However, the use of the substrate having a surface made of SiN is not particularly limited, and may be a measurement member used in an intermolecular interaction detection method other than RIfS, or other than an intermolecular interaction detection method. For example, a measuring member used in a measuring method using labeling such as a fluorescent label may be used. In addition, the substrate may be provided with an additional structure (for example, an electrode or the like) according to the method for detecting the interaction between molecules.

As for SiN on the substrate surface, an oxide film is formed from the moment when it is exposed to air. This oxide film has a functional group such as a hydroxyl group (hydroxyl group).
In the step (b), as the method for introducing the modifying group onto the substrate surface, for example, a method using a silane coupling agent having the modifying group or a polymer having the modifying group is applied using spin coating. Examples of the method include a method using a silane coupling agent from the viewpoint of ease of operation and prevention of the removal of the modifying group by covalently fixing to the substrate surface.

  Generally, by immersing a substrate in an aqueous solution of a silane coupling agent for a predetermined time, hydroxyl groups generated by oxidation in the air present on the surface made of SiN and silanol groups derived from the silane coupling agent And the like, and the silane coupling agent is bonded, and the surface made of SiN can be modified with a modifying group such as an epoxy group of the silane coupling agent.

  Further, in the step (c), the reaction between the modifying group and the reactive group tends to have a relatively long reaction time and slightly lower reactivity than the reaction using a conventional active esterification reagent. However, the reaction rate is constant.

  The epoxy group, azide group, isocyanate group or isothiocyanate group as the modifying group and the thiol group, amino group or hydroxyl group as the reactive group can be reacted without particularly requiring a reaction reagent. For example, by bringing an aqueous solution of a ligand having the thiol group or the like into contact with the substrate surface modified with the epoxy group or the like (for example, immersing the substrate in the aqueous solution for a certain period of time), the modified group and reaction Covalent bonds can be formed between groups.

  The ligand having a reactive group is preferably at least one selected from the group consisting of proteins, polypeptides, nucleic acids and sugars, and proteins are particularly preferable. Proteins and polypeptides have modifying groups such as thiol groups, amino groups, and hydroxyl groups at the terminals or side chains of the amino acids constituting them.

  On the other hand, nucleic acids and sugars (which may be monosaccharides, oligosaccharides or polysaccharides) have an amino group or a hydroxyl group in the molecular structure, but usually do not have a thiol group. Since an epoxy group is more reactive with a thiol group than an amino group or a hydroxyl group, when a nucleic acid or sugar is used as the ligand to react with an epoxy group, a thiol group must be introduced into the nucleic acid or sugar in advance. Is preferred.

(substrate)
There are two types of substrates manufactured in the present embodiment.
The first substrate is a modified group-introduced substrate, which is obtained by the method for producing a modified group-introduced substrate as described above. That is, the modified group-introduced substrate of the present embodiment has at least one modifying group selected from the group consisting of an epoxy group, an azide group, an isocyanate group, and an isothiocyanate group, preferably with a silane coupling agent on the surface made of SiN. The substrate is modified with This first substrate has good storage stability.

  The second substrate in the present embodiment is a ligand-immobilized substrate and is obtained by the above-described method for producing a ligand-immobilized substrate. That is, the ligand-immobilized substrate of the present embodiment has at least one modifying group selected from the group consisting of an epoxy group, an azide group, an isocyanate group, and an isothiocyanate group that has modified the surface of SiN. And a substrate on which the ligand is immobilized through a reaction with at least one reactive group selected from the group consisting of a thiol group, an amino group, and a hydroxyl group that the ligand had.

  However, the manufacturing method of the 1st and 2nd board | substrate of this invention is not limited to the manufacturing method of the 1st and 2nd board | substrate of this embodiment as mentioned above. Even if it is a case where it is obtained by a manufacturing method different from them, those having the same configuration as described above correspond to the first and second substrates of the present invention.

<Intermolecular interaction detection method>
The intermolecular interaction detection method of this embodiment is characterized by using the ligand-immobilized substrate.

(Configuration of detection device)
The intermolecular interaction detection method of the present embodiment can be carried out using an intermolecular interaction measuring apparatus for RIfS having a known general configuration. An outline of an example of an embodiment of a measurement system including such an intermolecular interaction measurement apparatus is shown in FIG.

  This measurement system 1 is mainly composed of an intermolecular interaction measuring device 100 composed of a measuring member 10, a white light source 20, a spectroscope 30, a light transmission unit 40, and the like, a control device 50, and the like. .

  The measurement member 10 is configured based on a sensor chip 12 including at least a substrate 12a and an optical thin film 12b formed on the substrate 12a. Usually, a flow cell 14 is further loaded on the sensor chip 12.

The substrate 12a is generally rectangular and is preferably made of, for example, Si [silicon], and the optical thin film 12b is preferably made of, for example, SiN.
The flow cell 14 is a transparent member made of, for example, silicone rubber (polydimethylsiloxane: PDMS). The flow cell 14 can be replaced with the sensor chip 12, and can be used in a disposable manner. A groove 14 a is formed in the flow cell 14. When the flow cell 14 is brought into close contact with the sensor chip 12, a sealed flow path 14b is formed. Both end portions of the groove 14a are exposed from the surface of the flow cell 14, one end portion is connected to the liquid feeding portion, and functions as an inlet 14c to which various solutions such as the sample solution 60 are supplied, and the other end The part is connected to the waste liquid part and functions as an outlet 14d for various solutions such as the sample solution 60.

  The light transmission unit 40 is a first optical fiber 41 serving as a first light transmission path for guiding white light from the white light source 20 to the measurement unit 200, and irradiation of white light from the first optical fiber 41. And a second optical fiber 42 as a second light transmission path for guiding the reflected light from the measurement unit 200 to the spectroscope 30. The end of the first optical fiber 41 on the white light source 20 side is connected to the connection port of the white light source 20. The optical fiber 41 connected to the connection port is arranged so that the light incident end face faces the halogen lamp 21. The end of the second optical fiber 42 on the spectroscope 30 side is connected to a connection port that receives light from the spectroscope 30.

  Each of the optical fibers 41 and 42 has a structure in which fine fibers are bundled. And the edge part by the side of the flow cell 14 of the 1st optical fiber 41 and the 2nd optical fiber 42 is faced compoundly so that each fine fiber may become one bundle. That is, the fine fibers constituting the first optical fiber 41 are distributed in the center on the end face on the flow cell 14 side, and the fine fibers constituting the second optical fiber 42 are bundles of fine fibers of the first optical fiber 41. It is distributed around it to surround it.

  The white light source 20 includes a halogen lamp and a housing that stores the halogen lamp. The housing is provided with a connection port for connecting the first optical fiber 41. In this embodiment, a white light source is used. However, the present invention is not limited to this, and any light source may be used as long as it emits light distributed over a wavelength range in which a change in the minimum reflectance wavelength described later can be detected.

  When the white light source 20 is turned on, the white light is irradiated onto the measurement unit 200 via the first optical fiber 41, and the reflected light is guided to the spectrometer 30 via the optical fiber 42. The spectroscope 30 detects the light intensity of the light at fixed wavelength intervals included in the light received by the light receiving unit, and outputs the light intensity to the control device 50 as the spectral intensity.

  In the present embodiment, reflected light from the measurement member 10 is received by the spectroscope 30. However, a light transmissive member is used as the measurement member 10, and light from the white light source 20 is measured by the measurement member. The spectroscope 30 may be disposed so as to receive the light that has been irradiated to the measurement member 10 and transmitted through the measurement member 10, and the spectral intensity of the transmitted light may be detected.

  In the present embodiment, reflected light from the measurement member 10 is received by the spectroscope 30. However, a light transmissive member is used as the measurement member 10, and light from the white light source 20 is measured by the measurement member. It is also possible to arrange the spectroscope 30 so as to receive the light that has been irradiated onto the measuring member 10 and transmitted through the measuring member 10, and to be modified so as to detect the spectral intensity of the transmitted light.

  The control device 50 is composed of, for example, a PC [Personal Computer], receives an input of execution of the detection operation from the operator, and outputs a detection operation control execution command to the intermolecular interaction measuring device 100. Thereby, the control apparatus 50 functions as a control part.

  The control device 50 also functions as a calculation unit. The control device 50 acquires the spectral intensity data of the measurement light from the spectroscope 30 and calculates the reflectance for each wavelength band by dividing the spectral intensity of the measurement light by the spectral intensity of the white light as a reference. The spectral intensity data of the reference light may be measured and held in advance at the time of device assembly adjustment, or may be acquired by other means, for example, every measurement. A reflection spectrum is created based on the calculated reflectance, and the reflectance minimum wavelength is determined.

  The waveform of the reflection spectrum usually has an irregular shape in which minute irregularities are repeated, and it may be difficult to calculate and specify the reflectance minimum wavelength. By approximating the reflection spectrum with a high-order function using a known method, the waveform is smoothed, and the solution (minimum value) is obtained from the high-order polynomial, and this can be specified as the value of the minimum reflectance wavelength. .

  Although it is possible to directly control the white light source 20, the spectroscope 30, and a temperature adjusting unit, which will be described later, by the control device 50, the white light source 20, the white light source 20, the spectroscope 30, etc. It is preferable to separately provide a control unit (not shown) including a microcomputer for controlling the operation of each unit such as the light source 20, the spectroscope 30, and the temperature adjusting means. In this case, the microcomputer performs control to switch on / off the white light source 20 according to the control command of the control device 50, or performs temperature control of the temperature control unit according to the set temperature command of the control device 50.

  The temperature adjustment means (not shown) includes, for example, a temperature adjustment element that performs heating and cooling, such as a Peltier element, and a temperature detection element, and these are attached to the measurement member 10. And the control apparatus 50 acquires the temperature information of the measurement member 10 from a temperature detection element directly or through a microcomputer, and performs temperature control directly or through a microcomputer so that it may become set temperature by the heating or cooling by a temperature control element. To do.

  When performing the detection, the measurement member 10 is warmed up in advance. That is, the control device 50 controls the temperature control unit so as to achieve a predetermined set temperature or sends a command to the microcomputer so as to achieve the predetermined set temperature, and the microcomputer controls the temperature of the temperature control unit. Run. The analysis is started after the temperature of the measuring member 10 is stabilized by the warm air.

  The control device 50 determines whether or not to continue the measurement, and if not, ends the process. For example, the measurement time may be set in advance, and it may be determined whether or not the measurement time has elapsed, or the measurement end input is set as a setting for continuing the measurement until the measurement end input is received. The presence or absence may be determined. When the measurement is continued, the spectral intensity is measured again. By repeating the measurement, the control device 50 periodically calculates the reflectance, creates a reflection spectrum and determines the minimum reflectance wavelength, and records the time-series change.

(Analysis items)
There are no particular limitations on the method of using various types of information that can be acquired by the intermolecular interaction detection method, such as what items are analyzed based on the acquired measurement values.

For example, in the analysis according to RIfS, when a substance to be measured is captured by a sensor chip and a layer is formed, the wavelength (interference wavelength) at which the spectral reflectance is minimized before and after the formation of the layer. From the amount of change (Δλ), the thickness (d ₁ ) of the layer made of the substance to be measured can be obtained. That is, when a layer made of a substance to be measured that can be optically regarded as a thin film is formed on the surface of the sensor chip and interference of reflected light occurs, the thickness of the layer made of the substance to be measured can be easily obtained by the following equation: .

d ₁ = Δλ / 2n
n is the refractive index of the analyte and is usually in the range of about 1.4 to 1.6.
For reference (preparation of calibration curve), the amount of change in interference wavelength (Δλ ′) is measured using a solution containing an analyte having a known concentration, or the thickness of the layer made of the analyte (d ₁ ′) Is calculated, the change in the interference wavelength (Δλ) is measured using a sample whose analyte concentration is unknown, or the thickness (d ₁ ) of the layer made of the analyte is calculated, and then compared. Thus, the concentration of the analyte in the sample can be quantified.

  When the analyte is captured on the surface of the sensor chip for RIfS and a layer made of the analyte is formed, the amount of capture or the thickness of the layer is the amount of change in the interference wavelength (Δλ) measured by RIfS. In addition to being reflected in the numerical value, it is also reflected in the color when the sensor chip is visually observed (the visible color changes depending on the amount of capture or the thickness of the layer).

Next, specific examples of the present invention will be described, but the present invention is not limited to these examples.
[Example 1]
(Process (a): Fabrication of SiN sensor chip)
On the silicon wafer, SiN was deposited as an optical thin film so as to have a thickness of 66.5 nm to produce a SiN sensor chip.

(Process (b): Modification of epoxy group on SiN sensor chip surface)
To 10 mL of ultrapure water, 0.1 mL of GPTS [(3-glycoxypropyl) trimethylsilane] as a silane coupling agent and 0.1 mL of acetic acid were added.

The SiN sensor chip produced in the step (a) was immersed in this silane coupling agent solution for 1 hour at room temperature.
The SiN sensor chip thus treated was washed with ethanol and ultrapure water, and then water droplets were removed by nitrogen blowing, followed by drying at 110 ° C. for 2 hours with a dryer (dehydration condensation). Thus, the epoxy group modification to the SiN sensor chip surface was performed.

(Step (c): Immobilization of ligand)
Anti-human α-fetoprotein [AFP] mouse monoclonal antibody (clone: 1D5; Mikuri Immuno Laboratory Co., Ltd.) was prepared as a ligand using 10 mM acetate buffer (pH 6.0) so as to be 10 μg / mL. . The sensor chip just obtained in the above step (b) was immersed in this antibody solution at room temperature overnight. Thereafter, the sensor chip was washed with ultrapure water, whereby the antibody as a ligand could be immobilized on the surface of the SiN sensor chip. The obtained sensor chip is hereinafter also referred to as “ligand-immobilized substrate”.

(Confirmation of ligand-immobilized substrate performance)
The RifS type intermolecular interaction measurement device (MI-Affinity; manufactured by Konica Minolta Opto) was turned on and waited for about 20 minutes until the light source was stabilized. Also, a ligand-immobilized substrate, a flow cell (manufactured by Konica Minolta Opto Co., Ltd.) having a groove having a width of 2.5 mm, a length of 16 mm, and a depth of 0.1 mm, and through-holes having a diameter of 1 mm at both ends of the groove, The liquid was allowed to pass over the sensor chip through the chip cover provided in the measuring device. A 10 × PBS (−) buffer (pH 7.4; Wako Pure Chemical Industries, Ltd .: Code No. 314-90185) was added to the ultrapure water from the outside of the measuring apparatus by a syringe pump (Econoflo 70-2205; manufactured by Harvard Apparatus). A solution prepared by adding 10% of surfactant Tween 20 (manufactured by GE Healthcare Japan Co., Ltd.) to a buffer prepared by diluting 10 times with a solution is fed at a flow rate of 20 μL / min for 20 minutes, and a measurement standard It was confirmed that the wavelength λ ₀ (baseline) at which the spectral reflectance becomes the minimum becomes stable in the vicinity of about 570 nm.

(Antigen-antibody reaction by the RIfS method)
After the measurement was started, the reagent was injected through the injector of the intermolecular interaction measurement device at the timing shown below. The composition of the reagent and the addition timing (shown in parentheses with the elapsed time from the start of measurement) are as follows.
1) PBS buffer (5 min) in which 1% BSA is dissolved for confirmation of non-specific adsorption;
2) 1 μg / mL AFP (20 min) dissolved in PBS buffer (pH 7.4) for antigen-antibody reaction;
3) 10 μg / mL AFP (30 min) dissolved in PBS buffer (pH 7.4) to confirm the presence or absence of concentration dependency for antigen-antibody reaction;
4) 10 mM Gly-HCl (pH 1.5) (40 min) as a regeneration buffer for dissociating the antigen
5) 1 μg / mL AFP (50 min) dissolved in PBS buffer (pH 7.4) to confirm the reproducibility of the antigen-antibody reaction;
6) 10 μg / mL AFP (60 min) dissolved in PBS buffer (pH 7.4) to confirm the reproducibility of concentration dependence;
That is, for the ligand-immobilized substrate for which measurement preparation was completed, it was confirmed in 1) that there was no non-specific adsorption through the injector provided in the measurement device (in FIG. 3 (a) described later, protein was added). After confirming the absence of non-specific adsorption based on the phenomenon that the baseline becomes almost constant after a temporary decrease in the measured value due to the influence of AFP, the antigen AFP was introduced in 2). Subsequently, 10-fold concentration AFP was introduced in 3). The bottom peak wavelength after introducing the antigen was λ ₁ . Thereafter, the regeneration buffer was introduced in 4) to dissociate the antigen from the antibody. Further, in order to confirm the reproducibility of the antigen-antibody reaction, AFP as an antigen was introduced in 5) at the same concentration as 2), and subsequently, 10-fold concentration AFP was introduced in 6).

The difference between λ ₁ and λ ₀ was Δλ, and the amount of change in bottom peak wavelength due to antigen introduction was measured. FIG. 3 (a) shows the measurement result of the change with time of Δλ after the start of measurement. In this way, by using a sensor chip modified with an epoxy group, the ligand can be immobilized simply by immersing it in a solution containing the ligand, and in FIG. As is clear from the comparison of the measurement results before and after the introduction, it can be understood that the results are very reproducible. Although the experiment was repeated several times in the same procedure, the same result could be obtained in all cases.

[Comparative Example 1]
In step (b) of Example 1, a ligand-immobilized substrate was prepared in the same manner as in Example 1 except that only 0.1 mL of acetic acid was added to 10 mL of ultrapure water without adding GPTS as a silane coupling agent. Manufactured and verified its performance. The measurement results are shown in FIG.
Since there was almost no difference between λ ₁ and λ ₀ after the antigen was injected, it can be understood that the ligand was not immobilized on the substrate.

[Example 2]
The measurement was performed in the same manner as in Example 1 except that the sensor chip with the modified epoxy group was left in the atmosphere for 24 hours and then the ligand was immobilized. Further, the measurement was performed in the same manner as in Example 1 except that the sensor chip modified with the epoxy group was left in the atmosphere for 1 week and then the ligand was immobilized. In any case, no particularly significant difference was found from the measurement result of Example 1, and it was found that the epoxy group-modified substrate was excellent in stability.

DESCRIPTION OF SYMBOLS 1 Measurement system 10 Measuring member 12 Sensor chip 12a Silicon substrate 12b SiN (silicon nitride) film 14 Flow cell 14a Groove 14b Sealed flow path 14c Inlet 14d Outlet 16 Ligand 20 White light source 30 Spectroscope 40 Light transmission part 41 First light Fiber 42 Second optical fiber 50 Control device 60 Sample solution 62 Analyte 100 Intermolecular interaction measuring device 200 Measuring unit

Claims (8)

Step (a): preparing a substrate having a surface made of SiN [silicon nitride];
Step (b): introducing at least one modifying group selected from the group consisting of an epoxy group, an azide group, an isocyanate group and an isothiocyanate group into the SiN surface of the substrate. A method for producing a modifying group-introducing substrate.   The manufacturing method of Claim 1 which implements the said process (b) using the silane coupling agent which has the said modification group.   The manufacturing method according to claim 1, wherein the substrate is a sensor chip for reflection interference spectroscopy (RIfS).   Step (c): a production method according to any one of claims 1 to 3, wherein a ligand having at least one reactive group selected from the group consisting of a thiol group, an amino group and a hydroxyl group is used as the reactive group. A method for producing a ligand-immobilized substrate, comprising the step of immobilizing on the surface of the modifying group-introducing substrate through a reaction with a modifying group possessed by the modifying group-introducing substrate obtained by the above step.   The production method according to claim 4, wherein the ligand is at least one selected from the group consisting of proteins, polypeptides, nucleic acids, and sugars.   A modification group-introducing substrate produced by the production method according to claim 1.   A ligand-immobilized substrate produced by the production method according to claim 4 or 5.   A medium containing an analyte is brought into contact with the surface on which the ligand of the ligand-immobilized substrate according to claim 7 is immobilized, and physical characteristics of the surface of the substrate in contact with the medium containing the analyte are measured. And detecting an intermolecular interaction.