WO2012096037A1

WO2012096037A1 - Method for detecting intermolecular interaction, and kit for use therein

Info

Publication number: WO2012096037A1
Application number: PCT/JP2011/072947
Authority: WO
Inventors: 義一栗原
Original assignee: コニカミノルタオプト株式会社
Priority date: 2011-01-12
Filing date: 2011-10-05
Publication date: 2012-07-19
Also published as: JPWO2012096037A1

Abstract

A method for detecting an intermolecular interaction is provided in which bottom wavelengths are easier to calculate and a larger change in bottom wavelength is obtained than in conventional methods. Also provided is a kit for use in the method. The method, which is for detecting an intermolecular interaction, is characterized by including: a step in which the wavelength (λ₀) corresponding to the bottom of spectral reflectance for the state in which a composite of a ligand with an analyte has not been formed is calculated; and a step in which the wavelength (λ₁) corresponding to the bottom of spectral reflectance for the state in which a composite of the ligand, a fluorescent colorant that has a maximum fluorescent wavelength (λ_f) on the shorter wavelength side than the bottom wavelength (λ₀), and the analyte has been formed is calculated.

Description

Method for detecting intermolecular interaction and kit used therefor

The present invention relates to a detection method for detecting an intermolecular interaction, particularly an intermolecular interaction such as a biomolecule or an organic polymer, and a kit used therefor.

Conventionally, measurement of binding such as intermolecular interaction between biomolecules such as antigen-antibody reaction and intermolecular interaction between organic macromolecules is generally performed by using a label such as radioactive substance or phosphor. I have been. This labeling takes time, and in particular, labeling a protein may involve a complicated method or the property of the protein may change depending on the labeling. Therefore, in recent years, as a means for directly detecting the binding between biomolecules and organic polymers without using a label, the RIFS method (Reflectometric Interference® Spectroscopy: reflection interference spectroscopy using the interference color change of an optical thin film). Method) has been proposed and put into practical use. The basic principle of the RIfS method is mentioned in Patent Document 1, Non-Patent Document 1, and the like.

The conventional RIfS method will be briefly described. In this method, as shown in FIG. 3, a substrate 102 provided with an optical thin film 104 is used. As shown in FIG. 3A, when the optical thin film 104 on the substrate 102 is irradiated with white light, the spectral intensity of the white light itself (reference light) is shown by a solid line 106 as shown in a typical example of FIG. The spectral intensity of the reflected light is represented by a solid line 108. At this time, n ₀ d = (2k−1) λ / 4, where n ₀ and d are the refractive index and thickness of the optical thin film 104, k is a natural number, and λ is the wavelength. ], The attenuation of the reflected light due to the interference becomes the largest at the wavelength λ satisfying the relational expression. When the reflectance is obtained from the spectral intensities of the irradiated white light and the reflected light, a reflection spectrum 110 having a bottom peak (minimum portion in the spectrum curve) represented by a solid line is obtained as shown in FIG.

In detecting the intermolecular interaction, as shown in FIG. 3B, a ligand 120 (a substance that can specifically bind to the substance to be analyzed by the intermolecular interaction) is provided on the optical thin film 104. When the ligand 120 is provided on the optical thin film 104, the optical thickness 112 at the site where the ligand 120 is provided changes, the optical path length changes, and the interference wavelength (detection intensity by the spectroscope becomes the smallest due to the reflection interference effect). Wavelength) also changes. That is, the position of the bottom peak of the spectral intensity distribution of the reflected light is shifted, and as a result, as shown in FIG. 5, the reflected spectrum 110 is shifted to the reflected spectrum 122 (see the dotted line portion). In this state, when the sample solution is caused to flow over the optical thin film 104, the ligand 120 and the analyte (analyte substance) 130 in the sample solution are bonded as shown in FIG. When the ligand 120 and the analyte 130 are bonded, the optical thickness 112 at the site where the analyte 130 is bonded further changes. The analyte 130 partially adheres to the ligand 120 to generate a heterogeneous layer. This macroscopic layer is macroscopically determined to have a predetermined optical thickness corresponding to the amount of the analyte 130 deposited. It is replaced with a homogeneous layer having a thickness. Accordingly, the optical thickness of the homogeneous layer through which incident light passes changes depending on the amount of the analyte 130 attached. As a result, as shown in FIG. 5, the reflection spectrum 122 is shifted to the reflection spectrum 132 (refer to the one-dot chain line portion). Then, by detecting the change Δλ _{1 ′} between the bottom peak wavelength λ _{0 of} the reflection spectrum 122 (the wavelength at which the reflectance becomes a minimum value) and the bottom peak wavelength λ _{1 ′} of the reflection spectrum 132, the ligand 120 and the analyte are detected. It is possible to detect the intermolecular interaction with 130, that is, the presence of the ligand 120 in the sample, and to detect the progress of the intermolecular interaction.

When the transition of the change in the bottom peak wavelength is observed over time, the change in the bottom peak wavelength due to the ligand 120 can be confirmed at the time point 140, which is the first shoulder portion on the curve, as shown in FIG. At the time 142, which is the second shoulder portion above, a change in the bottom peak wavelength due to the binding between the ligand 120 and the analyte 130 can be confirmed.

Japanese Patent No. 3778673

The conventional method is suitable for the measurement of the analyte 130 having a low concentration in the sample solution because the change Δλ _{1 ′} of the bottom peak wavelength caused by the presence of the analyte 130 is very small and the detection sensitivity is limited. Absent. Further, the data actually output by the detection device that detects the spectral intensity of the reflected light repeats minute fluctuations as shown in FIG. 7A, and this reflectance is used to determine the bottom peak position. An operation such as fitting an approximate curve to the data (see FIG. 7B) and obtaining the minimum value of the approximate curve as the above-described bottom peak position is required. Moreover, since the reflectance change is small near the bottom peak, in principle, the bottom peak position is likely to be inaccurate in the method of determining the bottom peak position using the approximate curve as described above. The peak wavelength change Δλ may change differently from the progress of intermolecular interaction. In such a case, the bottom peak wavelength change Δλ accurately indicates the progress of intermolecular interaction. There is a problem that it is no longer suitable as a value for judging.

An object of the present invention is to provide a method for detecting an intermolecular interaction that solves the above-described problems.

The inventor has developed a fluorescent dye that emits fluorescence having a spectral intensity distribution that satisfies a specific relationship with the spectral intensity distribution of reflected light in a state where a ligand is formed on the optical thin film (the state shown in FIG. 3B), In this method, the analyte 130 is labeled with a fluorescent dye having a maximum fluorescence wavelength λ _f on the shorter wavelength side than the bottom peak wavelength λ ₀ , and the spectrum of the measurement light including both the fluorescence and the reflected light is measured. Then, when the fluorescent dye is added, the spectrum of the fluorescent dye acts so as to exaggerate the change in the bottom peak wavelength, so that the change amount Δλ of the bottom peak wavelength λ ₁ of the spectrum from the bottom peak wavelength λ ₀ is increased. ₁ is easily observed than the conventional variation [Delta] [lambda] _{1 ',} found to be able to realize a method of detecting molecular interactions, such problems were solved as described above, thereby completing the present invention .

That is, the present invention includes the following embodiments.
[1]
A detection member having a ligand on the optical thin film; a light source that emits light distributed over a predetermined wavelength range; a spectroscope that detects a spectral intensity of the received light; and a light source that transmits light from the light source to the detection member. Using a detection device comprising one light transmission path and a light transmission unit having a second light transmission path for transmitting light from the light source from the detection member to the spectrometer,
Spectroscopy calculated by transmitting light from the light source to the detection member through the first and second light transmission paths, receiving light through the detection member, measuring the spectral intensity with the spectroscope, and the like. A method for detecting an intermolecular interaction between the ligand and the analyte by changing a bottom peak wavelength of an intensity distribution,
Calculating a bottom peak wavelength (λ ₀ ) of a spectral intensity distribution in a state where a complex of the ligand and the analyte is not formed; and in a wavelength region around the ligand and the bottom peak wavelength (λ ₀ ) A step of calculating the bottom peak wavelength (λ ₁ ) of the spectral intensity distribution in a state in which the complex of the fluorescent dye having the maximum fluorescence wavelength (λ _f ) and the analyte is formed so as to overlap the valley of the spectral intensity distribution A method for detecting an intermolecular interaction, comprising:

[2]
Item 2. The method for detecting an intermolecular interaction according to Item 1, wherein the complex is formed by binding an analyte labeled with the fluorescent dye to the ligand.

[3]
The detection of an intermolecular interaction according to Item 1, wherein the complex is formed by binding of the analyte to the ligand and further binding of the ligand labeled with the fluorescent dye to the analyte. Method.

[4]
Item 4. The detection of an intermolecular interaction according to any one of Items 1 to 3, wherein the fluorescent dye has a maximum fluorescence wavelength (λ _f ) on a shorter wavelength side than the bottom peak wavelength (λ ₀ ). Method.

[5]
The difference Δλ _f (= λ ₀ −λ _f ) between the maximum fluorescence wavelength (λ _f ) of the fluorescent dye and the bottom peak wavelength (λ ₀ ) satisfies a relationship of 0 nm <Δλ _f ≦ 100 nm. Method for detecting intermolecular interactions.

[6]
Item 6. The method for detecting an intermolecular interaction according to any one of Items 1 to 5, comprising at least a component for producing a detection member having the ligand and the fluorescent dye. Kit used for.

In the present invention, when an analyte is present by using a fluorescent dye having a maximum fluorescence wavelength (λ _f ) so as to overlap a valley of a spectral intensity distribution in a wavelength region around the bottom peak wavelength (λ ₀ ). The bottom peak wavelength of the observed spectral intensity distribution shifts to the longer wavelength side than before, that is, the amount of change in the bottom peak wavelength becomes larger than before. Further, the valley portion of the spectral intensity distribution graph observed when the analyte is present becomes steep, and the bottom peak wavelength can be easily specified. As a result, even if the concentration of the analyte is low, it can be detected with higher sensitivity than before, and quantitative analysis can be easily performed.

It is drawing which shows schematic structure of the detection apparatus of an intermolecular interaction. It is drawing which shows schematic structure of a detection member. It is drawing which demonstrated the outline of the RIfS system in order. In particular, (C) shows the state seen in the conventional mode, and (D) and (E) show the state seen in the first and second modes of the present embodiment, respectively. It is an example of a spectrum which shows the rough relationship between a wavelength and spectral intensity. It is an example of a spectrum which shows the rough relationship between the wavelength by the conventional method, and a reflectance. It is a graph which shows the rough transition of the change of a bottom peak wavelength. (A) is a diagram of a reflection spectrum obtained by detection, and (B) is a diagram of a higher-order function approximating the reflection spectrum. It is an example of a spectrum which shows the rough relationship between the wavelength by the method of this embodiment, and a reflectance.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.
(Configuration of intermolecular interaction detector)
FIG. 1 is a schematic diagram schematically illustrating the configuration of an intermolecular interaction detection apparatus 1 to which an intermolecular interaction detection method according to an embodiment of the present invention can be applied.

As shown in FIG. 1, the detection apparatus 1 mainly includes a detection member 10, a white light source 20, a spectroscope 30, a light transmission unit 40, a control device 50, and the like.
The detection member 10 basically includes a sensor chip 12 and a flow cell 14.

As shown in FIG. 2, the sensor chip 12 has a silicon substrate 12a having a rectangular shape. A SiN (silicon nitride) film 12b is deposited on the silicon substrate 12a. The SiN film 12b is an example of the optical thin film 104.

The flow cell 14 is a transparent member made of silicone rubber. 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 channel 14b is formed (see FIG. 1). 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 60 such as a sample solution are supplied, and the other end portion Is connected to the waste liquid section and functions as an outlet 14d for various solutions 60 such as sample solutions. The ligand 16 that binds to the analyte 62 is bound to the bottom of the groove 14a of the flow cell 14, that is, the surface of the sensor chip 12 (see FIG. 1).

In the detection member 10, the flow cell 14 can be replaced with respect to the sensor chip 12, and the flow cell 14 can be used in a disposable manner. The surface of the sensor chip 12 may be modified with a silane coupling agent or the like. In this case, the flow cell 14 can be easily replaced.

The light transmission unit 40 includes a first optical fiber 41 as a first light transmission path for guiding white light from the white light source 20 to the closed flow path 14 b of the flow cell 14, and white light from the first optical fiber 41. And a second optical fiber 42 as a second light transmission path for guiding reflected light by light irradiation from the sealed flow path 14b of the flow cell 1 to the spectroscope 30. As shown in FIG. 1, the end surfaces of the first and second optical fibers 41 and 42 are disposed in close contact with each other on the upper surface of the flow cell 14 and above the sealed flow path 14b.

The white light source 20 is composed of a halogen lamp and a housing for storing the halogen lamp. The housing is provided with a connection port for connecting the first optical fiber 41. The white light emitted from the white light source 20 includes excitation light of a fluorescent dye described later. 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 bottom peak wavelength described later can be detected.

The first optical fiber 41 is for guiding the white light emitted from the white light source 20 to the flow cell 14, and when the white light source 20 is turned on, the white light is sealed through the first optical fiber 41. Irradiate the path 14b. The end of the first optical fiber 41 on the white light source 20 side is connected to a 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 second optical fiber 42 is for guiding light from the flow cell 14 to the spectroscope 30, and the measurement light when the white light of the white light source 20 irradiates the sealed flow path 14 b is guided to the spectroscope 30 and detected. It is possible to do. 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. 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, the reflected light from the detection member 10 is received by the spectroscope 30. However, a light transmissive member is used as the detection member 10, and the light from the white light source 20 is detected by the detection member. The spectroscope 30 may be arranged so as to receive the light that has been irradiated to the light 10 and transmitted through the detection member 10, and the spectral intensity of the transmitted light may be detected.

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 control device 50 is composed of, for example, a PC (Personal Computer), receives an input of detection operation execution from an operator, and outputs a detection operation control execution command to the detection device 10. Thereby, the control apparatus 50 functions as a control part.

Further, the control device 50 receives the detection data of the spectral intensity of the white light serving as a reference and the spectral intensity of the reflected light based on the measurement from the spectroscope 30, and calculates the reflectance for each wavelength band based on these, Calculate the reflection spectrum. Thereby, the control apparatus 50 functions as a calculation part.

A microcomputer (not shown) performs control to switch on / off the white light source 20 according to a control command of the control device 50, or performs temperature control of a temperature control unit according to a set temperature command of the control device 50. To do.

The temperature control unit (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 provided together with the detection member 10. And the control apparatus 50 detects the temperature of the detection member 10 with a temperature detection element through a microcomputer, and performs temperature control so that it may become preset temperature by the heating or cooling by a temperature control element.

When detecting, the detection member 10 is warmed up in advance. That is, the control device 50 sends a command to the microcomputer so that the preset temperature is set in advance, and the microcomputer performs temperature control of the temperature control unit. After the temperature of the detection member 10 is stabilized by warm air, liquid feeding of the sample solution or the like is started.

When measuring the spectral intensity, the first optical fiber 41 guides the white light of the white light source 20 to the sealed flow path 14b of the flow cell 14 and irradiates it. Then, the measurement light is received by the spectrometer 30 through the second optical fiber 42, and the spectral intensity is detected. This spectral intensity is transmitted to the control device 50.

Next, the control device 50 determines whether or not to continue the measurement. If not, the process is terminated. 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 measurement of the spectral intensity with respect to the measurement light is performed again.

The control device 50 acquires the spectral intensity data of the measurement light. The control device 50 reflects each wavelength by dividing the light intensity of the measurement light by the measurement intensity of the reference light for each wavelength band by the spectral intensity of the white light (reference light) and the spectral intensity of the measurement light. The reflectance spectrum can be calculated by obtaining the rate. The spectral intensity data of the reference light may be measured and held in advance when the apparatus is assembled and adjusted, or may be acquired by other means. The control device 50 periodically acquires spectral intensity data of measurement light by repeated measurement. Thus, the reflection spectrum is periodically calculated, the bottom peak wavelength is further calculated, and the time-series change is recorded.

According to this embodiment, as will be described later, since it becomes relatively easy to determine the bottom peak wavelength from the reflection spectrum, the calculation for determining the bottom peak wavelength can be simplified. In order to increase, a correction calculation as described below may be used.

In other words, when the reflection spectrum 72 is calculated from the spectral intensity data of the reference light and the spectral intensity data of the measurement light, the waveform has an irregular shape in which minute irregularities are repeated, and the bottom peak wavelength is calculated and specified. In some cases, it is difficult to do this (FIG. 7A). At this time, for example, when the reflection spectrum 72 is approximated by a high-order function, the waveform of the reflection spectrum 72 can be made smooth as shown in FIG. The approximation may be performed by any known method. And the solution (minimum value) is calculated | required from this higher order polynomial, and this can be specified as a value of a bottom peak wavelength.

(Method for detecting intermolecular interactions)
In the detection method of the intermolecular interaction of this embodiment,
Step 1: calculating a bottom peak wavelength (λ ₀ ) of spectral reflectance in a state where the complex of the ligand and the analyte is not formed;
Step 2: Spectral reflectance in a state where a complex of the ligand, a fluorescent dye having a maximum fluorescence wavelength (λ _f ) on the shorter wavelength side than the bottom peak wavelength (λ ₀ ), and an analyte is formed. Calculating a bottom peak wavelength (λ ₁ ).

The step 2 includes two different modes to which the fluorescent dye is to be bound. In the first embodiment (Step 2A), as shown in FIG. 3D, the complex is bound to the ligand 120 with the analyte 130 (fluorescently labeled analyte 230) labeled with the fluorescent dye 200. To be formed. In the second embodiment (step 2B), as shown in FIG. 3E, after the analyte 130 is bound to the ligand 120, the complex is further labeled with the fluorescent dye 200. The ligand 120 (fluorescently labeled ligand 240) is formed by binding.

More specifically, the measurement of the reflection spectrum and the calculation of the bottom peak wavelength performed periodically as described above are step 1 in which λ ₀ is obtained as follows, and in the first embodiment, λ _{1 is} as follows: Step A for obtaining λ _1A , and in the second embodiment, λ ₁ is performed so as to include Step B for obtaining λ _1B as described below.

(Step 1: λ ₀ acquisition step)
In a state where the ligand 120 is bonded to the surface of the optical thin film 104, an aqueous solution (for example, an appropriate buffer solution) that does not contain the fluorescently labeled analyte 230, the fluorescently labeled ligand 240, or the like from the liquid feeding part to the sealed flow path 14b of the flow cell 14. ) And is circulated from the inlet 14c to the outlet 14d through the sealed channel 14b. Thus, in the state where the sealed channel 14b is filled with the aqueous solution, the spectral intensity data of the measurement light is acquired by the means as described above. The calculated bottom peak wavelength is λ ₀ .

(Process 2A: λ _1A acquisition process)
In the first aspect, after the λ ₀ acquisition step, an aqueous solution containing the fluorescently labeled analyte 230 is supplied from the liquid feeding unit to the sealed flow path 14b of the flow cell 14, and the flow outlet 14c passes through the closed flow path 14b. 14d. Then, the fluorescently labeled analyte 230 is captured by the immobilized ligand 120, and the optical thickness 112 becomes thicker than in the λ ₀ acquisition step. In such a state, the spectral intensity data of the measurement light is obtained by the means as described above, the calculated bottom peak wavelength is λ _1A , and the difference between the bottom peak wavelength λ _1A and the bottom peak wavelength λ ₀ is The amount of change in the bottom peak wavelength is Δλ _1A .

(Process 2B: λ _1B acquisition process)
In the second aspect, after the λ ₀ acquisition step, the aqueous solution containing the analyte 130 is supplied from the liquid feeding section to the sealed flow path 14b of the flow cell 14, and the inlet 14c passes through the sealed flow path 14b to the outlet 14d. Distributed. Then, the analyte 130 is captured by the immobilized ligand 120. Subsequently, an aqueous solution containing the fluorescently labeled ligand 240 is supplied from the liquid feeding section to the sealed flow path 14b of the flow cell 14, and is circulated from the inlet 14c to the outlet 14d via the sealed path 14b. Then, the fluorescently labeled ligand 240 further binds to the analyte 130 bound to the immobilized ligand 120, and the optical thickness 112 is larger than that in the λ ₀ acquisition step (and more than in the λ _1A acquisition step). Become thicker. In such a state, the spectral intensity data of the measurement light is acquired by the means as described above. The calculated bottom peak wavelength is λ _1B , and the difference between the bottom peak wavelength λ _1B and the bottom peak wavelength λ ₀ is the change amount Δλ _1B of the bottom peak wavelength.

(Typical change in bottom peak wavelength)
A typical example of the spectral spectrum 81 obtained in step 1 and the spectral spectrum 82 obtained in step 2 are shown in FIG. As shown in FIG. 8, the spectral spectrum 82 obtained in Step 2 is different from the spectral spectrum 81 having the valley obtained in Step 1 in that the valley is shifted to the long wavelength side and the valley of the spectral spectrum 82 is obtained. A shoulder portion S derived from the fluorescent dye is formed on the short wavelength side. In FIG. 8, the spectral intensity of the fluorescent dye is indicated by a dotted line, and the maximum fluorescent wavelength of the fluorescent dye overlaps with the valley in the spectral intensity distribution in the wavelength region around the bottom peak wavelength λ ₀ . By forming the shoulder portion S, the bottom of the valley of the spectrum 82 is pushed to the longer wavelength side as a whole, and the bottom peak wavelength λ ₁ of the spectrum 82 is the case where no fluorescent dye is included. Compared to the bottom peak wavelength λ _{1 ′} in the spectrum after the analyte coupling, the bottom peak wavelength moves to the longer wavelength side, and the shift width of the bottom peak wavelength becomes large. Further, by forming the shoulder portion S, the valley portion of the spectral spectrum 82 becomes steeper than the valley portion of the spectral spectrum 81, and the reflectance at the bottom peak wavelength is lower in the region where the reflectance is lower than the shoulder portion S. When the difference in reflectance (h1 and h2 in FIG. 8) becomes the same, the width w2 of the valley of the spectrum 82 is narrower than the width w1 of the valley of the spectrum 81. Therefore, it is easy to specify the bottom peak wavelength λ ₁ after the analyte coupling, and it is easy to grasp the amount of change in the bottom peak wavelength. For this reason, the calculation for specifying the bottom peak wavelength is also facilitated.

Fluorescent dye The fluorescent dye used in this embodiment is a spectral intensity distribution that satisfies a specific relationship with the spectral intensity distribution of reflected light in the state of FIG. 3B when irradiated with predetermined excitation light (incident light). And fluorescent dyes (fluorescent molecules or fluorophores) that emit fluorescence. More specifically, a fluorescent dye having a maximum fluorescence wavelength (λ _f ) is used so as to overlap a valley portion of the spectral intensity distribution in a wavelength region around the bottom peak wavelength (λ ₀ ) described above.

Typically, a fluorescent dye having a maximum fluorescence wavelength (λ _f ) on the shorter wavelength side than the bottom peak wavelength (λ ₀ ) is used. The difference Δλ _f (= λ ₀ −λ _f ) between the maximum fluorescence wavelength (λ _f ) and the bottom peak wavelength (λ ₀ ) of this fluorescent dye has a relationship of ₀ nm <Δλ _f (= λ ₀ −λ _f ) ≦ 100 nm. It is preferable to satisfy, and it is more preferable to satisfy the relationship of 0 nm <Δλ _f ≦ 50 nm. In this case, the maximum fluorescence wavelength λ _f of an appropriate fluorescent dye is the bottom peak wavelength λ ₀ acquired as data in advance, or the bottom peak wavelength expected from the thickness and refractive index of the optical thin film 104 (for example, the SiN film 12b). It can be set by taking λ ₀ into consideration.

On the other hand, according to the study of the present inventors, the maximum fluorescence wavelength of the fluorescent dye is not necessarily must be in a shorter wavelength than the bottom peak wavelength lambda _0, the bottom peak wavelength lambda ₀ same or bottom peak wavelength and lambda ₀ It may be on the long wavelength side within a predetermined range. Also in this case, the bottom peak wavelength λ ₁ can be shifted to a longer wavelength side than the bottom peak wavelength λ _{1 ′} when no fluorescent dye is used. In this case, the absolute value of the difference Δλ _f between the maximum fluorescence wavelength (λ _f ) and the bottom peak wavelength (λ ₀ ) of the fluorescent dye is preferably within 80 nm. Maximum fluorescence wavelength of the fluorescent dye, detailed reason for the bottom peak wavelength lambda ₁ even in the long wavelength side within a predetermined range than or equal bottom peak wavelength lambda ₀ and the bottom peak wavelength lambda ₀ is shifted to the long wavelength side is not available However, if the maximum fluorescence wavelength of the fluorescent dye is close to the bottom peak wavelength λ ₀ to some extent, the short wavelength component of the fluorescent dye contributes to the same spectral spectrum deformation as described above. .

Fluorescent dyes such as those described above can be selected from among general fluorescent dyes used in various measurement systems (fluorescent labeling method, etc.) that satisfy a specific target condition. Examples of fluorescent dye candidates include fluorescent dyes of the fluorescein family (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan Co., Ltd.), and hexachlorofluorescein family fluorescent dyes. Dye (Applied Biosystems Japan), Coumarin Family Fluorescent Dye (Invitrogen), Rhodamine Family Fluorescent Dye (GE Healthcare Biosciences), Cyanine Family Fluorescent Dye, Indian Carbo Cyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide family fluorescent dye, BODIPY (registered trademark) Family fluorescent dyes (Invitrogen Corporation), naphthalenesulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, Alexa Fluor (registered trademark) dye series (Invitrogen Corporation) And organic fluorescent dyes.

In addition to the organic fluorescent dyes described above, rare earth complex fluorescent dyes such as Eu and Tb (for example, ATBTA-Eu ³⁺ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), and green fluorescent protein. (GFP), yellow fluorescent protein (YFP), red fluorescent protein (DsRed), fluorescent protein typified by Allophycocyanin (APC; LyoFlogen (registered trademark)), fluorescent fine particles such as latex and silica can also be used Can be a candidate for a dye.

In particular, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594 Cy3, a fluorescent dye selected from the fluorescein family, and a rhodamine family, GFP It is preferable.

Analyte and Ligand The analyte 130 is a substance to be analyzed in which a substance that specifically binds to the analyte 130 (that is, a substance that becomes the ligand 120) exists. For example, biomolecules such as proteins (including polypeptides, oligopeptides, etc.), nucleic acids (including DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc.), lipids, sugars, drug substances, endocrine disruptions A foreign substance or the like that binds to a biomolecule such as a chemical substance can be used as the analyte 130. On the other hand, as the ligand 120, an appropriate substance that specifically binds to the analyte 130 is selected. For example, if the analyte 130 is a protein or peptide serving as an antigen, the protein serving as its antibody is a nucleic acid having a complementary base sequence if the analyte 130 is a nucleic acid, and if the analyte 130 is a sugar, A binding lectin (protein) or the like is used as the ligand 120.

Fluorescently labeled analyte and fluorescently labeled ligand In the first embodiment, “fluorescently labeled analyte” 230 that is the analyte 130 to which the fluorescent dye 200 is linked is used. On the other hand, in the second embodiment, a “fluorescently labeled ligand” 240 that is the ligand 120 to which the fluorescent dye 200 is linked is used. The ligand of the fluorescently labeled ligand 240 may be the same as or different from the ligand of the immobilized ligand 120 as long as an immobilized ligand-analyte-fluorescently labeled ligand complex can be formed. For example, when the immobilized ligand 120 is a polyclonal antibody, the fluorescently labeled ligand 240 may be a monoclonal antibody or a polyclonal antibody, but when the immobilized ligand 120 is a monoclonal antibody, the fluorescently labeled ligand 240 is It is desirable that the antibody is a monoclonal antibody that recognizes an epitope that the immobilized ligand does not recognize, or a polyclonal antibody.

The fluorescently labeled analyte 230 and the fluorescently labeled ligand 240 can be produced according to a known method. For example, when the analyte 130 or the ligand 120 is a protein or a nucleic acid, the functional group possessed by the selected fluorescent dye 200 and the functional group possessed by the analyte 130 or ligand 120 are directly used using a predetermined reagent. Alternatively, the fluorescence-labeled analyte 230 or the fluorescence-labeled ligand 240 is obtained by binding indirectly (through another molecule such as a linker).

When the fluorescently labeled analyte 230 is used in the first aspect, before starting the λ _1A acquisition step, for example, the collected sample is prepared by reacting with a predetermined reagent inside or outside the detection apparatus 1. An aqueous solution containing the fluorescently labeled analyte 230 is prepared. On the other hand, when the fluorescently labeled ligand 240 is used in the second embodiment, before starting the λ _1B acquisition step, for example, the fluorescently labeled ligand 240 is set in the detection apparatus 1 as a separately prepared reagent. Prepare an aqueous solution containing it.

Sensor chip The optical thin film 104 constituting the sensor chip 12 is formed of a material having a refractive index and a thickness such that a bottom peak observed when white light is used is in an appropriate range. For example, the optical thin film 104 is preferably a SiN film 12b. The refractive index of SiN is about 2.0 to 2.5 at a wavelength range of about 400 to 800 nm in the visible region. By setting the thickness of the SiN film to about 45 to 90 nm, the bottom peak is about 400 nm to 800 nm. Can be adjusted to the range.

Further, it is necessary to modify the surface of the sensor chip 12 so that the ligand 120 corresponding to the analyte 130 is bonded prior to the start of the λ ₀ acquisition step. For example, the surface of the unmodified SiN film 12b is treated with a silane coupling agent and modified with an amino group, followed by treatment with NHS (N-hydroxysuccinimide) -PEG4-biotin, and biotin is added to the amino group. After binding and reacting this biotin with avidin, a biotinylated antibody or nucleic acid is reacted to produce a sensor chip 12 having an antibody or nucleic acid as the ligand 120 on the surface. Such a modification can be performed by setting the sensor chip 12 having an unmodified surface in the detection apparatus 1 and then sequentially feeding the reagent and the cleaning liquid for the modification as described above.

Kit As a detection kit used in the intermolecular interaction detection apparatus of the present embodiment, a sensor chip 12 that is a component for producing the detection member 10 having the ligand 120 and a fluorescent dye 200 are set. If it prepares, it will become easy to use for those who want to detect. In the first aspect described above, the fluorescently labeled analyte 230 may be set instead of the fluorescent dye 200. In the second aspect described above, the fluorescently labeled ligand 240 is set instead of the fluorescent dye 200. You may do it. In any case, since a suitable combination of the fluorescent dye 200 and the sensor chip 12 is set, the user does not get lost in the combination of the fluorescent dye 200 and the sensor chip 12.

[Example 1]
(Step 1: SiN sensor chip surface amino group modification)
On the silicon wafer (100), silicon nitride (SiN) as an optical thin film was CVD-deposited to a thickness of 66.5 nm to produce a SiN sensor chip.

100 μl of 3-APTES ((3-Aminopropyl) triethoxysilane: manufactured by Shin-Etsu Chemical Co., Ltd.) was gradually dropped into a mixed solution of 9.5 ml ethanol and 0.5 ml of ultrapure water, and the mixture was allowed to stand at room temperature for 1 hour. Stir. The SiN sensor chip was immersed therein and further stirred at room temperature for 1 hour.

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 80 ° C. for 1 hour with a dryer. In this way, amino group modification was performed on the surface of the SiN sensor chip.

(Step 2: Surface biotin modification of SiN sensor chip)
NHS-PEG4-Biotin (ThermoFisher Scientific KK) was prepared with 10 ml of 10 mM sodium borate buffer (pH 8.5) so as to be 200 μg / ml, and the sensor chip modified with amino group in step 1 was prepared. It was immersed for 1 hour at room temperature to introduce biotin onto the surface.

(Step 3: Preparation for RIfS measurement)
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. In addition, the sensor chip manufactured in step 2, a groove having a width of 2.5 mm, a length of 16 mm, and a depth of 0.1 mm, and a flow cell having a through hole having a diameter of 1 mm at both ends of the groove (Konica Minolta Opto Co., Ltd.) 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), PBS buffer (pH 7.4; manufactured by Nacalai Tesque Co., Ltd.) is fed from the outside of the measuring apparatus at a flow rate of 20 μl / min for 20 minutes, and spectral reflection as a measurement standard It was confirmed that the wavelength λ ₀ (baseline) at which the rate is minimum is stable in the vicinity of about 570 nm.

(Step 4: Antigen-antibody reaction between primary antibody and antigen by RIfS method)
A sample prepared by adjusting NeutrAvidin (manufactured by ThermoFisher Scientific KK) to 100 μg / ml using the PBS buffer is passed through the injector provided in the measuring device to the sensor chip that has been prepared for measurement in step 3. Introduced. Subsequently, a sample prepared by using a biotinylated anti-α-fetoprotein (AFP) antibody (clone 1D5; manufactured by Mikuli Immuno Laboratory Co., Ltd.) as a primary antibody at 10 μg / ml using the PBS buffer was introduced. Subsequently, AFP (manufactured by Acris Antibodies GmbH) as an antigen and a sample prepared at 10 μg / ml using the PBS buffer were introduced. The bottom peak wavelength after introducing the antigen was λ _{1 ′} .

(Step 5: Antigen-antibody reaction between antigen and secondary antibody by RIfS method)
Subsequent to step 4, various anti-AFP antibodies (clone 6D2; manufactured by Mikuri Immuno Laboratory Co., Ltd.) are used as secondary antibodies using Alexa Fluor 488 Monoclonal Antibody Labeling Kit (manufactured by Invitrogen, maximum fluorescence wavelength λ _f = 515 nm). Four samples of antibodies modified with a fluorescent dye were prepared. Each sample prepared to 10 μg / ml using the above PBS buffer was introduced into the prepared antibody. The bottom peak wavelength after the introduction of the fluorochrome-labeled secondary antibody was lambda _1. The amount of change in the bottom peak wavelength due to the introduction of the secondary antibody was measured with Δλ being the difference between λ ₁ and λ ₀ .

[Example 2]
The antigen-antibody reaction was detected by the RIfS method in the same manner as in Example 1 except that the step 5 ′ was changed to the step 5 ′.
(Step 5 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
Subsequent to step 4, various anti-AFP antibodies (clone 6D2; manufactured by Mikuli Immuno Laboratory Co., Ltd.) are used as secondary antibodies using Alexa Fluor 555 Monoclonal Antibody Labeling Kit (manufactured by Invitrogen, maximum fluorescence wavelength λ _f = 565 nm). Four samples of antibodies modified with a fluorescent dye were prepared. Each sample prepared to 10 μg / ml using the above PBS buffer was introduced into the prepared antibody. The bottom peak wavelength after the introduction of the fluorochrome-labeled secondary antibody was lambda _1. The amount of change in the bottom peak wavelength due to the introduction of the secondary antibody was measured with Δλ being the difference between λ ₁ and λ ₀ .

[Example 3]
The antigen-antibody reaction was detected by the RIfS method in the same manner as in Example 1 except that the step 5 ′ was changed to the step 5 ′.
(Step 5 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
Subsequent to step 4, various anti-AFP antibodies (clone 6D2; manufactured by Mikuli Immuno Laboratory Co., Ltd.) are used as secondary antibodies using Alexa Fluor 594 Monoclonal Antibody Labeling Kit (manufactured by Invitrogen, maximum fluorescence wavelength λ _f = 617 nm). Four samples of antibodies modified with a fluorescent dye were prepared. Each sample prepared to 10 μg / ml using the above PBS buffer was introduced into the prepared antibody. The bottom peak wavelength after the introduction of the fluorochrome-labeled secondary antibody was lambda _1. The amount of change in the bottom peak wavelength due to the introduction of the secondary antibody was measured with Δλ being the difference between λ ₁ and λ ₀ .
[Example 4]

The antigen-antibody reaction was detected by the RIfS method in the same manner as in Example 1 except that the step 5 ′ was changed to the step 5 ′.
(Step 5 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
Subsequent to step 4, various anti-AFP antibodies (clone 6D2; manufactured by Mikuli Immuno Laboratory Co., Ltd.) are used as secondary antibodies using Alexa Fluor 647 Monoclonal Antibody Labeling Kit (manufactured by Invitrogen, maximum fluorescence wavelength λ _f = 665 nm). Four samples of antibodies modified with a fluorescent dye were prepared. Each sample prepared to 10 μg / ml using the above PBS buffer was introduced into the prepared antibody. The bottom peak wavelength after the introduction of the fluorochrome-labeled secondary antibody was lambda _1. The amount of change in the bottom peak wavelength due to the introduction of the secondary antibody was measured with Δλ being the difference between λ ₁ and λ ₀ .

[Comparative Example 1]
When step 4 was completed, the bottom peak wavelength change amount (difference between λ _{1 ′} and λ ₀ ) Δλ ′ due to the conventional antigen-antibody reaction alone was measured.

[Comparative Example 2]
The antigen-antibody reaction was detected by the RIfS method in the same manner as in Example 1 except that the step 5 ′ was changed to the step 5 ′.

(Step 5 ': Antigen-antibody reaction of antigen and secondary antibody by RIfS method)
Subsequent to step 4, an anti-AFP antibody (clone 6D2; manufactured by Mikuli Immuno Laboratory Co., Ltd.) as a secondary antibody was introduced to a concentration of 10 μg / ml using the PBS buffer. The bottom peak wavelength after the introduction of the secondary antibody with lambda _1. The amount of change in the bottom peak wavelength due to the introduction of the secondary antibody was measured with Δλ being the difference between λ ₁ and λ ₀ .

The value of Δλ (λ ₁ -λ ₀ ) in Examples 1 to 4 and Comparative Example 2, the value of Δλ ′ (λ _{1 ′} -λ ₀ ) in Comparative Example 1, and the bottom peak measured using the RIfS method As an indicator of the stability of the detection value of the wavelength, the former is followed by the secondary antibody introduction at the time of λ ₁ detection, and the latter is introduced after the antigen introduction at the time of λ _{1 ′} detection, after the reaction reaches an equilibrium state. Table 1 shows the results (baseline stability) of calculating the standard deviation of the 50 continuously detected bottom peak wavelength values.

From the results of Table 1, the difference between the maximum fluorescence wavelength (λ _f ) and the bottom peak wavelength (λ ₀ ) of the fluorescent dye is 0 nm with respect to the RIfS bottom peak wavelength (570 nm) in a state where no ligand is attached to SiN. <By labeling with Alexa 488 and Alexa 555 satisfying the relational expression of <(λ ₀ −λ _f ) ≦ 100 nm, the RIfS system measures the spectrum of the measurement light including both fluorescence and reflected light. Acts to push the reflection spectrum of the measurement object to the longer wavelength side, and as a result, the change in the bottom peak wavelength is exaggerated and Δλ is observed to be large (Example 1, Example 2). In particular, a 555 nm fluorescent dye closest to the bottom peak wavelength of SiN is most effective. Moreover, since the spectrum near the bottom peak of the reflectance of the fluorescent dye becomes steep, the bottom peak wavelength becomes stable and easy to detect, so that the stability of the baseline is also expected to increase. On the other hand, when the maximum fluorescence wavelength (lambda _f) was used fluorescent dye with the longer wavelength side than the bottom peak wavelength (lambda _0), the maximum fluorescence wavelength (lambda _f) is a bottom peak wavelength (lambda ₀₎ shorter than Although not as much as when a fluorescent dye having a wavelength is used, the change width Δλ of the bottom peak wavelength is larger than that when no fluorescent dye is used. Of course, it is clear that this is more effective than detecting only the reaction with the antigen against the immobilized antibody as shown in Comparative Example 1, which is a conventional method of using RIfS.

DESCRIPTION OF SYMBOLS 1 Detection apparatus 10 Detection member 12 Sensor chip 12a Silicon substrate 12b SiN (silicon nitride) film | membrane 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 controller 60 aqueous solution 62 analyte 100 detection member 102 substrate 104 optical thin film 112 optical thickness 120 ligand 130 analyte 200 fluorescent dye 230 fluorescent label analyte 240 fluorescent label ligand 106 spectral intensity (reference) light)
108 Spectral intensity (reflected light)
110 Reflection spectrum (state of FIG. 3A)
122 Reflection spectrum (state of FIG. 3B)
132 Reflection spectrum (state of FIG. 3C)
140 First shoulder portion 142 Second shoulder portion 72 Reflection spectrum 81 Spectral spectrum obtained in step 1 82 Spectral spectrum obtained in step 2

Claims

A detection member having a ligand on the optical thin film; a light source that emits light distributed over a predetermined wavelength range; a spectroscope that detects a spectral intensity of the received light; and a light source that transmits light from the light source to the detection member. Using a detection device comprising one light transmission path and a light transmission unit having a second light transmission path for transmitting light from the light source from the detection member to the spectrometer,
Spectroscopy calculated by transmitting light from the light source to the detection member through the first and second light transmission paths, receiving light through the detection member, measuring the spectral intensity with the spectroscope, and the like. A method for detecting an intermolecular interaction between the ligand and the analyte by changing a bottom peak wavelength of an intensity distribution,
Calculating a bottom peak wavelength (λ 0 ) of a spectral intensity distribution in a state where a complex of the ligand and the analyte is not formed; and in a wavelength region around the ligand and the bottom peak wavelength (λ 0 ) A step of calculating the bottom peak wavelength (λ 1 ) of the spectral intensity distribution in a state in which the complex of the fluorescent dye having the maximum fluorescence wavelength (λ f ) and the analyte is formed so as to overlap the valley of the spectral intensity distribution A method for detecting an intermolecular interaction, comprising:
The method for detecting an intermolecular interaction according to claim 1, wherein the complex is formed by binding an analyte labeled with the fluorescent dye to the ligand.
The detection of an intermolecular interaction according to claim 1, wherein the complex is formed by binding of the analyte to the ligand and further binding of the ligand labeled with the fluorescent dye to the analyte. Method.
The detection of an intermolecular interaction according to any one of claims 1 to 3, wherein the fluorescent dye has a maximum fluorescence wavelength (λ f ) on a shorter wavelength side than the bottom peak wavelength (λ 0 ). Method.
The difference Δλ f (= λ 0 −λ f ) between the maximum fluorescence wavelength (λ f ) and the bottom peak wavelength (λ 0 ) of the fluorescent dye satisfies the relationship of 0 nm <Δλ f ≦ 100 nm. Method for detecting intermolecular interactions.
The method for detecting an intermolecular interaction according to any one of claims 1 to 5, comprising at least a constituent member for producing a detection member having the ligand and the fluorescent dye. Kit used for.