JP2013011464A - Ligand immobilization method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a substrate to which ligands are highly densely connected, even when a conventional ligand immobilization method is applied to a substrate whose one surface comprises SiN, TaO, NbO, HfO, ZrOor ITO, a ligand immobilization substrate obtained by the manufacturing method, and an intermolecular interaction detecting method using the substrate.SOLUTION: The manufacturing method for ligand immobilization substrates includes a step for immobilizing ligands in a liquid phase on the surface of a substrate comprising at least one type selected from a group comprising SiN, TaO, NbO, HfO, ZrOand ITO. The manufacturing method is characterized in that a surfactant contained in the liquid phase is not less than 0 mass% and less than 0.001 mass%.

Description

  The present invention relates to a method for producing a substrate suitable as a sensor chip for reflection interference spectroscopy (RIfS), a substrate obtained by the production method, and a molecular 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. 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, an antibody that can specifically bind to the surface of a sensor chip that uses a certain antigen as a substance to be measured is immobilized as a capture substance.

  As described above, there are many methods for immobilizing a ligand on a sensor chip. For example, in a microchannel system of a measurement device “Biacore” (GE Healthcare) for SPR, Examples thereof include amide coupling in which a carboxyl group that modifies the substrate and an amino group of the ligand are reacted. In this method, the manufacturer recommends using a solvent called HBS-EP containing Surfactant 20 (Tween 20) as a surfactant.

However, when this method was applied to a substrate whose surface was composed of SiN, Ta ₂ O ₅ , Nb ₂ O ₅ , HfO ₂ , ZrO ₂ or ITO, the ligand did not bind to the substrate at all.

The present invention is a case where a conventional ligand immobilization method is applied to the surface of a substrate having at least one surface thereof made of SiN, Ta ₂ O ₅ , Nb ₂ O ₅ , HfO ₂ , ZrO ₂ and / or ITO. However, it is an object of the present invention to provide a method for manufacturing a substrate in which a ligand is bonded to the substrate at a high density, a substrate obtained by the manufacturing method, and a method for detecting an intermolecular interaction using the substrate.

The inventor includes a surfactant when a conventional ligand immobilization method is applied to the surface of a substrate made of SiN, Ta ₂ O ₅ , Nb ₂ O ₅ , HfO ₂ , ZrO ₂ and / or ITO. When a solvent not containing a surfactant was used instead of the solvent recommended by the manufacturer, it was found that the ligand was covalently bonded to the substrate at a high density, and the present invention was completed.

That is, the present invention includes the following matters.
[1] SiN [silicon nitride], Ta ₂ O ₅ [tantalum oxide], Nb ₂ O ₅ [niobium pentoxide], HfO ₂ [hafnium oxide], ZrO ₂ [zirconium dioxide] and ITO [indium tin oxide] A method for producing a ligand-immobilized substrate, comprising a step of immobilizing a ligand in a liquid phase on the surface of at least one substrate selected from the group,
A production method, wherein the surfactant contained in the liquid phase is 0% by mass or more and less than 0.001% by mass.

  [2] In the step of immobilizing the ligand, the ligand is immobilized by reacting the modifying group on the surface of the substrate with the reactive group on the ligand to form a covalent bond [1]. The manufacturing method as described in.

[3] The production method according to [1] or [2], wherein the modifying group on the surface of the substrate is introduced using a silane coupling agent.
[4] The production method according to any one of [1] to [3], wherein the ligand is at least one selected from the group consisting of proteins, polypeptides, nucleic acids, and sugars.

  [5] The substrate is a sensor chip for reflection interference spectroscopy (RifS), surface plasmon resonance (SPR), or crystal oscillator microbalance (QCM) [1] to [4] ] The manufacturing method in any one of.

[6] A ligand-immobilized substrate produced by the production method according to any one of [1] to [5].
[7] A method for detecting an intermolecular interaction, comprising using the ligand-immobilized substrate according to [6].

According to the present invention, when a conventional ligand immobilization method is applied to the surface of a substrate made of SiN, Ta ₂ O ₅ , Nb ₂ O ₅ , HfO ₂ , ZrO ₂ and / or ITO, the surfactant is added. By using a solvent that does not contain, or a solvent that contains a very small amount of a surfactant, the ligand can be covalently bonded to the substrate at high density.

FIG. 1 schematically shows a reaction for immobilizing a ligand via a carboxyl group on the surface of a SiN substrate (preferably a sensor chip). FIG. 2 shows the result (a) of Example 1, the result (b) of Example 2, and the result (c) of Comparative Example 1. FIG. 3 is a schematic diagram of an example of an embodiment of the present invention used for RIfS.

Next, embodiments of the method for producing a ligand-immobilized substrate of the present invention, the substrate obtained by the production 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.

<Method for producing ligand-immobilized substrate and substrate>
The substrate used in the method for producing a ligand-immobilized substrate according to the present invention is SiN [silicon nitride], Ta ₂ O ₅ [tantalum oxide], Nb ₂ O ₅ [niobium pentoxide], HfO ₂ [hafnium oxide], ZrO _2. It has at least one surface selected from the group consisting of [zirconium dioxide] and ITO [indium tin oxide].

  When the surface of the substrate is SiN, an oxide film is formed from the moment when it is exposed to air. This oxide film has at least a functional group such as a hydroxyl group (hydroxyl group). Therefore, it is possible to introduce a modifying group using a silane coupling agent as described later.

  The method for producing a ligand-immobilized substrate according to the present invention includes a step of immobilizing a ligand in the liquid phase on the surface of the predetermined substrate, and the surfactant contained in the liquid phase includes: It is characterized by being 0% by mass or more and less than 0.001% by mass, preferably 0% by mass or more and less than 0.0001% by mass, and most preferably 0% by mass (that is, no surfactant is contained). The lower the surfactant concentration, the higher the ligand immobilization rate.

  Surfactants include ionic surfactants, including anionic (anionic) surfactants, cationic (cationic) surfactants and amphoteric surfactants, and nonionic (nonionic) surfactants. The In the present invention, for example, a nonionic surfactant is suitable.

  Nonionic surfactants include those of an ester ether type, an ester type, an ether type, etc. Among them, an ester ether type is preferable. Examples of ester ether type nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tokyo Chemical Industry Co., Ltd., trade name “Tween 20”), polyoxyethylene sorbitan monopalmitate (“Tween 40”), poly Examples thereof include oxyethylene sorbitan monostearate (same “Tween 60”), polyoxyethylene sorbitan monooleate (same “Tween 80”), and polyoxyethylene sorbitan trioleate (same “Tween 85”).

Any one of the surfactants exemplified above may be used alone, or two or more thereof may be mixed and used within a range not impairing the effects of the present invention.
The liquid phase is generally a buffer (eg, a sodium acetate buffer containing acetic acid and sodium acetate or a phosphate buffered saline containing phosphate, sodium phosphate and salt) or pure water, There is added an amount of a surfactant that provides a concentration in the above predetermined range.

  In the step of immobilizing the ligand in the production method of the present invention, various methods can be employed to immobilize the ligand on the surface of the substrate-like measurement member (sensor chip or the like). As a typical method, there is a method of immobilizing a ligand by introducing a modifying group on the surface of the substrate and reacting with a reactive group of the ligand to generate a covalent bond. In order to use such a method, in the present invention, at least the surface selected from the group consisting of an amino group, a carboxyl group, an epoxy group, an aldehyde group, a thiol group, an isocyanate group, and an isothiocyanate group is previously formed on the surface of the substrate. It is preferable to introduce one type of modifying group. These modifying groups react with thiol groups, amino groups, carboxyl groups, hydroxyl groups, etc. that the ligand has (which may be inherent to the ligand or previously introduced by a predetermined operation) to form a covalent bond. be able to.

  Examples of the method for introducing the modifying group onto the substrate surface include a method using a silane coupling agent having the modifying group and a method of applying a polymer having the modifying group using spin coating. A method using a silane coupling agent is preferable from the viewpoint of ease of operation and the effect of preventing the elimination of the modifying group by immobilization on the substrate surface by a covalent bond.

  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 The surface of SiN can be modified with a modifying group possessed by the silane coupling agent and the silane coupling agent.

  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, carboxyl groups, and hydroxyl groups at the ends 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 amino groups or hydroxyl groups in their molecular structures, but usually do not have carboxyl groups or thiol groups. If desired, these reactive groups may be introduced into nucleic acids or sugars. For example, among the above-mentioned modifying groups, an epoxy group has a higher reactivity with a thiol group than an amino group or a hydroxyl group. It is preferable to introduce a group.

  Appropriate combinations of the above-mentioned modifying groups and reactive groups, and methods for reacting with the combinations can be selected. For example, when a carboxyl group is selected as the modifying group and an amino group is selected as the reactive group, NHS [N-hydroxysuccinimide] and EDC (1-ethyl-3- (3) are first formed on the substrate surface modified with the carboxyl group. -Dimethylaminopropyl) Carbodiimide hydrochloride (sometimes called Water Soluble Carbodiimide: WSC). A covalent bond can be formed between the active esterified carboxyl group and the amino group. In addition, when an epoxy group, azide group, isocyanate group or isothiocyanate group is selected as a modifying group, and a thiol group, amino group or hydroxyl group is selected as a reactive group, the above epoxy group or the like is not particularly required without a reaction reagent. By contacting an aqueous solution of a ligand having the thiol group or the like with the substrate surface modified with (for example, immersing the substrate in the aqueous solution for a certain period of time), the covalent bond is formed between the modifying group and the reactive group. Can be formed. The reaction conditions (reaction time, reaction temperature, pH, etc.) at that time may be adjusted to an appropriate range.

  The ligand-immobilized substrate manufactured by the manufacturing method of the present invention is suitable for a sensor chip for reflection interference spectroscopy [RIfS], for surface plasmon resonance [SPR] or for quartz oscillator microbalance method [QCM]. It can also be applied to other sensor chips. However, the use of the ligand-immobilized substrate is not particularly limited, and may be a measurement member used in a method for detecting intermolecular interactions other than RIfS, or a method other than the method for detecting intermolecular interactions, For example, it may be a measuring member used in a measuring method using labeling such as a fluorescent label.

<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]
(Step 1: Modification of carboxyl group on SiN sensor chip surface)
To an unmodified SiN sensor chip (manufactured by Konica Minolta Opto Co., Ltd.), 100 μL of triethoxysilylpropylameric acid (manufactured by Gelest, Inc.) is gradually dropped into 10 mL of 1% acetic acid aqueous solution and stirred at room temperature for 1 hour. did. The SiN sensor chip was immersed therein and further stirred at room temperature for 1 hour.

  After the SiN sensor chip thus treated was washed with ultrapure water, water droplets were removed with a blower, and then dried at 80 ° C. for 1 hour with a dry heat sterilizer (STA620DA; manufactured by Advantech Co., Ltd.). In this way, the carboxyl group was modified on the surface of the SiN sensor chip.

(Step 2: Preparation for RIfS measurement)
The intermolecular interaction measuring apparatus (MI-Affinity; manufactured by Konica Minolta Opto Co., Ltd.) adopting RIfS as the measurement principle was turned on and waited for about 20 minutes until the light source was stabilized. In addition, the sensor chip manufactured in step 1, a PDMS-made groove 2.5 mm wide × 16 mm long × 0.1 mm deep, and a flow cell (Konica Minolta Opto, respectively) having through-holes 1 mm in diameter 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. With a syringe pump (Econoflo 70-2205; manufactured by Harvard Apparatus, Inc.), PBS buffer (pH 7.4) is fed from the outside of the measuring apparatus at a flow rate of 20 μl / min for 20 minutes, and the spectral reflectance serving as a measurement standard is minimized. It was confirmed that the wavelength (baseline) becomes stable at around 570 nm.

(Step 3: antibody immobilization using RIfS method)
In step 2, the measurement was started for the sensor chip for which measurement preparation was completed. The wavelength at which the spectral reflectance at the measurement start time is minimized is λ ₀ . Measure the 25 mM MES buffer (pH 5.0) through the injector provided in the above measuring device so that it becomes 50 mM NHS (manufactured by ThermoFisher Scientific KK), 200 mM WSC (manufactured by Dojindo Laboratories). It was introduced 300 seconds after the start, and the esterification of the carboxyl group-modified sensor chip was carried out. Subsequently, a sample prepared by preparing an anti-α-fetoprotein [AFP] antibody (clone 1D5; manufactured by Mikuli Immuno Laboratory Co., Ltd.) to 10 μg / mL using 10 mM sodium acetate buffer (pH 6.0) from the start of measurement was 1500. After 2 seconds, 2700 seconds later, the antibody was immobilized twice to immobilize the antibody. Finally, 1M ethanolamine-HCl (pH 8.5) was introduced after 3900 seconds from the start of measurement as blocking of the active ester. The bottom peak wavelength after the introduction of ethanolamine was λ ₁ .
The measurement result of the value of Δλ (λ ₁ -λ ₀ ) by antibody immobilization is shown in Table 1, and the RIfS measurement result of antibody immobilization in real time is shown in FIG.

[Example 2]
The antibody was immobilized in the same manner as in Example 1 except that Step 2 in Example 1 was replaced with Step 2 ′ below. The measurement results of Δλ (λ ₁ -λ ₀ ) values obtained by antibody immobilization are shown in Table 1, and the RifS measurement results of antibody immobilization in real time are shown in FIG.

(Step 2 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
The intermolecular interaction measuring apparatus (MI-Affinity; manufactured by Konica Minolta Opto Co., Ltd.) adopting RIfS as the measurement principle was turned on and waited for about 20 minutes until the light source was stabilized. In addition, the sensor chip manufactured in step 1, a PDMS-made groove 2.5 mm wide × 16 mm long × 0.1 mm deep, and a flow cell (Konica Minolta Opto, respectively) having through-holes 1 mm in diameter 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. Using a syringe pump (Econoflo 70-2205; manufactured by Harvard Apparatus, Inc.), a solution obtained by adding 0.0005 mass% of nonionic surfactant Tween 20 to PBS buffer (pH 7.4) from the outside of the measuring apparatus at a flow rate of 20 μl / min. The solution was fed for 20 minutes, and it was confirmed that the wavelength (baseline) at which the spectral reflectance serving as a measurement reference was minimum was stabilized at about 570 nm.
That is, Step 2 ′ is different from Step 2 only in that a predetermined concentration of surfactant is added to the PBS buffer.

[Comparative Example 1]
The antibody was immobilized in the same manner as in Example 1 except that Step 2 in Example 1 was replaced with Step 2 ′ below. Table 1 shows the measurement results of Δλ (λ ₁ -λ ₀ ) values obtained by antibody immobilization, and FIG.

(Step 2 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
The intermolecular interaction measuring apparatus (MI-Affinity; manufactured by Konica Minolta Opto Co., Ltd.) adopting RIfS as the measurement principle was turned on and waited for about 20 minutes until the light source was stabilized. In addition, the sensor chip manufactured in step 1, a PDMS-made groove 2.5 mm wide × 16 mm long × 0.1 mm deep, and a flow cell (Konica Minolta Opto, respectively) having through-holes 1 mm in diameter 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. Using a syringe pump (Econoflo 70-2205; manufactured by Harvard Apparatus, Inc.), a solution obtained by adding 0.001% by mass of nonionic surfactant Tween 20 to PBS buffer (pH 7.4) from the outside of the measuring apparatus at a flow rate of 20 μl / min. The solution was fed for 20 minutes, and it was confirmed that the wavelength (baseline) at which the spectral reflectance serving as a measurement reference was minimum was stabilized at about 570 nm.

  That is, step 2 'is different from step 2 only in that a predetermined concentration of surfactant is added to the PBS buffer.

From these results, it was found that using a solution containing less than 0.001% by mass of a surfactant on the SiN surface is more suitable for immobilizing the ligand.

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 (7)

Selected from the group consisting of SiN [silicon nitride], Ta ₂ O ₅ [tantalum oxide], Nb ₂ O ₅ [niobium pentoxide], HfO ₂ [hafnium oxide], ZrO ₂ [zirconium dioxide] and ITO [indium tin oxide]. A method for producing a ligand-immobilized substrate, comprising a step of immobilizing a ligand in a liquid phase on the surface of at least one kind of substrate,
A production method, wherein the surfactant contained in the liquid phase is 0% by mass or more and less than 0.001% by mass.   2. The ligand according to claim 1, wherein in the step of immobilizing the ligand, the ligand is immobilized by reacting a modifying group on the surface of the substrate with a reactive group on the ligand to form a covalent bond. Production method.   The method according to claim 1 or 2, wherein the modifying group on the surface of the substrate is introduced using a silane coupling agent.   The production method according to any one of claims 1 to 3, wherein the ligand is at least one selected from the group consisting of proteins, polypeptides, nucleic acids, and sugars.   5. The sensor chip according to claim 1, wherein the substrate is a sensor chip for reflection interference spectroscopy (RIfS), surface plasmon resonance (SPR), or quartz crystal microbalance (QCM). The production method according to item.   A ligand-immobilized substrate produced by the production method according to claim 1.   A method for detecting an intermolecular interaction, comprising using the ligand-immobilized substrate according to claim 6.