JP2013029372A - Ionic functional group modification sensor chip, and intermolecular interaction measurement method using ligand-carrying charged fine particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method with higher sensitivity that solves problems that measurement methods using magnetic particles and magnetic force have while making good use of advantage obtained by combining a homogeneous assay and heterogeneous sensing (intermolecular interaction measurement method) with each other.SOLUTION: There is provided an intermolecular interaction measurement method that uses a sensor chip having a positively- or negatively-charged layer as a surface layer, the intermolecular interaction measurement method including the processes of: forming a ligand-carrying charged fine particle-analyte composite by subjecting positively- or negatively-charged fine particles charged opposite to the charged layer, ligand-carrying charged fine particles composed of ligands bonded to the charged fine particles, and an analyte to reaction in a medium; and bringing a medium including the ligand-carrying charged fine particle-analyte composite into contact with a surface of the charged layer, and observing a signal generated by an act of intermolecular interaction.

Description

  The present invention relates to a sensor chip for a non-labeled intermolecular interaction measurement method such as RIFS (Reflectometric Interference Spectroscopy), and an intermolecular interaction measurement method using the sensor chip.

  In recent years, non-labeled intermolecular interaction measurement methods, that is, measuring members that emit a specific signal when intermolecular interactions such as biomolecules and organic polymers work without using labels such as radioactive substances and phosphors. Research and development of a method for directly and quantitatively detecting a measurement object by observing the signal using a (sensor chip) is underway. For example, RIfS (Reflectometric Interference Spectroscopy), which is a detection method that uses the interference color change of an optical thin film, has been proposed and put into practical use. In addition, intermolecular interaction measurement methods such as QCM (Quartz Crystal Microbalance) are also known.

  In the sensor chip for these intermolecular interaction measuring devices, a capture substance (ligand) that has a specific intermolecular interaction with a substance to be detected (analyte) is immobilized on the surface layer. For example, when a certain protein (antigen) is used as an analyte, a sensor chip in which a protein (antibody) that can specifically bind to the protein is immobilized on the surface as a ligand is used. When these analytes and ligands are bound by an intermolecular interaction, that is, an antigen-antibody reaction, the state of the sensor chip changes, and a signal resulting therefrom can be observed.

  Such intermolecular interaction measurement methods are generally heterogeneous, in which an analyte-containing medium (liquid phase) is brought into contact with a substrate-like sensor chip (solid phase) on which a ligand is immobilized. Sensing. Although this measurement method is highly sensitive, the reaction between the ligand and the analyte occurs only at the interface between the substrate-like solid phase and the liquid phase. There are big restrictions.

  On the other hand, the homogeneous assay in which the analyte in the liquid phase is brought into contact with the ligand supported on the fine particles dispersed in the liquid phase even in the solid phase is compared with the heterogeneous contact. The reaction efficiency is good, and there is an advantage that there is an acceleration means (for example, stirring, vortexing, shaking, ultrasonic waves, etc.) for further increasing the reaction efficiency.

As a measurement method combining the heterogeneous reaction and the homogeneous reaction as described above, a method using magnetic particles and magnetic force is known.
For example, in Patent Document 1, in the method of quantitatively measuring an antigen or antibody in a liquid medium by the following steps (a) to (c), the intensity of transmitted light or scattered light accompanying the reaction in step (a) A method for measuring an antigen or antibody, characterized in that is measured:
(A) an antigen or antibody to be measured, a first reagent containing insoluble magnetic particles carrying the antigen or antibody against the antigen or the antibody, and an insoluble label carrying the antibody or antigen against the antigen or the antibody Reacting a second reagent containing particles in a liquid medium;
(B) Separating unreacted insoluble magnetic particles and agglomerated particles containing insoluble magnetic particles from the reaction mixture by applying a magnetic field to the reaction mixture of step (a), then the liquid medium and unreacted insoluble fluorescent labeled particles Removing
(C) A step of quantitatively measuring the antigen or antibody by quantifying the amount of the insoluble labeled particles reacted with the remaining insoluble magnetic particles.

JP 2000-146975 A (Patent No. 3695178)

  In the measurement method using magnetic particles as described in Patent Document 1 described above, the smaller the size of the magnetic particles, the larger the total surface area of the magnetic particles contained in the unit volume, that is, the magnetic particles. It is considered that the amount of the ligand supported on the surface of is large, and therefore the reaction efficiency with the analyte is high. However, if the size of the magnetic particles is below a certain level (for example, about 1 μm or less), there arises a problem that the magnetic flux cannot be collected well.

  The present invention has solved the problems found in the measurement method using magnetic particles and magnetic force, while taking advantage of the combination of homogeneous assay and heterogeneous sensing (intermolecular interaction measurement method). An object is to provide a sensitive measurement method.

  First, the inventors can introduce ionic functional groups having a positive or negative charge at a high density by, for example, coating an ionic polymer on the surface of the sensor chip for the intermolecular interaction measurement method. I found what I could do. In addition, the ligand-carrying charged fine particles in which the ligand is supported on charged fine particles having a charge opposite to that of the ionic functional group and the analyte are reacted in the liquid phase (homogeneous reaction system). After forming the charged fine particle-analyte complex, the aqueous solution containing the complex is brought into contact with the sensor chip modified with the ionic functional group, and bonded to the ionic functional group by electrostatic interaction. Thus, the present inventors have found that the complex can be captured on the surface of the sensor chip at a uniform and high density, and thus signals can be observed with higher sensitivity and higher accuracy than in the past, and the present invention has been completed.

That is, the present invention includes the following matters.
[1] A ligand-carrying charged fine particle, which is composed of positively or negatively charged charged fine particles and a ligand bonded thereto.

[2] The ligand-carrying charged fine particles according to [1], wherein the charged fine particles are made of SiO ₂ , TiO ₂ , an ionic polymer, or a metal capable of fixing a ligand.
[3] The intermolecular interaction measurement method using a sensor chip including a charged layer that is positively or negatively charged on a surface layer, wherein the charge is opposite to the charged layer, according to [1] or [2] A step of reacting a ligand-carrying charged fine particle and an analyte in a medium to form a ligand-carrying charged fine particle-analyte complex; and a medium containing the ligand-carrying charged fine particle-analyte complex, the surface of the charged layer A method for measuring an intermolecular interaction, comprising the step of observing a signal appearing when the intermolecular interaction is brought into contact with the substrate.

[4] The intermolecular interaction measuring method according to [3], wherein the charged layer is a layer made of an ionic polymer.
[5] The intermolecular interaction measurement method according to [3] or [4], wherein the ionic polymer is a polymer having a cationic functional group.

[6] The intermolecular interaction measurement method according to any one of [3] to [5], wherein a surface charge density of the charged layer is in a range of 50 to 200 mC / m ² .
[7] The intermolecular interaction measurement method according to any one of [3] to [6], wherein the arithmetic average roughness of the surface of the charged layer is in the range of 1 to 50 nm.

  [8] The intermolecular interaction measurement method according to any one of [3] to [7], wherein the sensor chip is for RIfS or QCM.

  According to the present invention, the charge possessed by the ionic functional group provided on the surface of the sensor chip captures the ligand-carrying charged fine particle-analyte complex having the opposite charge in a uniform and high density (in close-packed state). can do. By using such an intermolecular interaction measurement method according to the present invention, analyte detection and quantification can be performed with higher sensitivity and higher accuracy than in the past.

FIG. 1 is a schematic diagram of an example of an embodiment of the present invention in an aspect according to RIfS. FIG. 2 is an atomic force microscope (AFM) image of the surface of the cationically modified sensor chip prepared in Preparation Example 1.

  In the present invention, the “molecular interaction measurement method” refers to an intermolecular relationship between a predetermined molecule (ligand) immobilized on the surface of a measurement member (sensor chip) and a molecule to be detected (analyte). It is a general term for the method of detecting the analyte that caused the intermolecular interaction by observing the signal that appears when the interaction is activated. However, a method for observing a signal based on fluorescence or radiation generated by a label such as an enzyme, fluorescent substance, or radioisotope for producing a fluorescent dye as the signal is excluded. Representative examples of the method for measuring intermolecular interaction in the present invention include RIfS and QCM. Hereinafter, the present invention will be described in more detail with a focus on embodiments of RIfS. However, the method for measuring intermolecular interaction in the present invention is not limited to RIfS. A person skilled in the art can develop the present invention in a method for measuring intermolecular interactions other than QCM and other RIfS based on the description in the present specification.

-Ionic functional group modified sensor chip-
In the present invention, an ionic functional group-modified sensor chip in which a positively or negatively charged layer (charged layer) is formed on the surface, that is, the surface is modified with an ionic functional group is used. That is, the charged fine particle-ligand-analyte complex (including medium) is brought into contact with the surface of the ionic functional group-modified sensor chip, and measurement for detecting an intermolecular interaction is performed.

(Ionic functional group)
The ionic functional group of the charged layer includes a cationic functional group and an anionic functional group. When the sensor chip is used in combination with anionic charged fine particles, a cationic functional group is selected as the ionic functional group, and conversely, when used in combination with cationic charged fine particles, the ion An anionic functional group is selected as the functional functional group. Each of the cationic functional group and the anionic functional group may be one kind alone, or two or more kinds may be mixed. In the charged layer, usually only one of a cationic functional group or an anionic functional group is present, but a charged state that does not hinder the capture of charged fine particles can be generated. Both of these may be present. In addition, the charged state of the charged layer (ionic functional group) and the charged state of the charged fine particles used in combination therewith may vary depending on the surrounding pH where they are placed. It should be adjusted to an appropriate range.

Cationic functional group Examples of the cationic functional group include guanidinium ion, -NH ₃ ⁺ , -NH ₂ (CH ₃ ) ⁺ , -NH (CH ₃ ) ₂ ⁺ , -N (CH ₃ ) ₃ ⁺ And quaternary ammonium cations. Such cationic functional groups are preferably those that form an inner salt, such as halogen anions such as chlorine and bromine, sulfate anions, sulfonate anions, phosphate anions, carboxylate anions and the like. It may combine to form a counter salt. The cationic functional group is preferably a quaternary ammonium cation from the viewpoint of surface charge density. Although there is no restriction | limiting in particular as a polymer to contain the said cationic functional group, From the viewpoint of sensor performance, the film forming property is good and the thing which is not water-soluble is preferable.

· The anionic functional group anionic functional group, for example, -CO ₂ ^- (carboxylic acid groups), ^- SO ₃ - (sulfonic acid groups), ^- OSO ₃ - (sulfate group), ^- OPO ₄ - (phosphoric acid Group), -B (OH) ₂ (boronic acid) and the like. Such an anionic functional group preferably forms an inner salt, but may be bonded to a metal cation or an organic cation. Examples of the metal cation include alkali metals such as Na ⁺ (sodium cation), K ⁺ (potassium cation), and Ca ⁺ (calcium cation). Examples of the organic cation include Me ₃ N ^+. H (trimethylammonium cation), Et ₃ N ⁺ H (triethylammonium cation), Me ₂ N ⁺ H ₂ (dimethylammonium cation) and the like can be mentioned.

In addition, said ionic functional group (A) may be couple | bonded directly with the polymer used as a principal chain, and may couple | bond with the polymer principal chain through another coupling group R (AR). -Polymer main chain). Examples of the bonding group -R- include an alkylene group having 1 to 6 carbon atoms, a phenylene group, and an ethyleneoxy group ((C ₂ H ₄ O) _n ).

(Method for forming charged layer)
A sensor chip having a charged layer on the outermost surface can be produced by forming a charged layer on the surface of an unmodified sensor chip by the method described below. The “unmodified sensor chip” may be prepared according to the method for measuring the interaction between molecules. As described above, an unmodified sensor chip for RIfS is generally composed of a substrate (for example, Si) and an optical thin film (for example, SiN) formed on the surface layer side thereof. An unmodified sensor chip for QCM is generally composed of an AT-cut crystal resonator and an electrode (for example, Au) formed on the surface layer side thereof.

  The method for forming the charged layer is not particularly limited. For example, the charged layer is made of a polymer having an ionic functional group in the side chain (ionic polymer) from the viewpoint of surface charge strength and durability. A method of forming a film on the surface of the sensor chip is preferable.

-Ionic polymer The ionic polymer may be obtained by previously polymerizing a monomer having an ionic functional group, or after polymerizing a monomer having another functional group capable of inducing or introducing an ionic functional group. Further, it may be obtained by inducing or introducing an ionic functional group into the functional group.

  One of the ionic polymers includes those having a hydrocarbon skeleton as the main chain. Examples of the monomer used for synthesizing such an ionic polymer include monomers having a vinyl group, an allyl group, a diene, and the like. More specifically, for example, amides such as (meth) acrylamide; (meth) acrylic acid or carboxylic acids such as vinyl formate, vinyl acetate, allyl acetate, allyl acetoacetate, vinylmaleic acid or esters thereof; styrene Sulfonic acid or sulfonic acid such as styrene sulfonic acid ester or esters thereof; in addition, sulfuric acid ester, phosphoric acid ester, phosphonic acid ester and the like are also included.

  Substances known as urethane resins having an ionic functional group introduced therein, for example, cationic aqueous polyurethane resins (for example, “Hydran (registered trademark) CP” series, DIC Corporation) can also be used in the present invention. It is mentioned as an ionic polymer.

  Furthermore, an ionic polymer having a backbone derived from a polyamino acid as the main chain may also be mentioned. Examples of such ionic polymers include polyaspartic acid and polyglutamic acid having a carboxyl group in the side chain, and polylysine having an amino group in the side chain.

  The various ionic polymers described above can be synthesized by known methods, and can also be obtained as commercial products. The properties of the ionic polymer, such as the number of ionic functional groups per molecule and the number average molecular weight, can be appropriately adjusted according to the charge state of the target sensor chip surface.

  The ionic polymer can be applied to the surface of the sensor chip using a known method such as dip coating, spin coating, spray coating or the like. When using such a coating method, an ionic polymer may be dissolved in an appropriate solvent so as to have an appropriate concentration to prepare a coating solution, which may be applied. In general, the concentration of the ionic polymer in the coating solution is 0.5 to 10% by weight, and the coating film thickness is adjusted in the range of 5 nm to 500 nm for the RIfS sensor chip and 5 nm to 100 nm for others. A charged layer made of a conductive polymer can be formed.

  If necessary, a SAM (Self-Assembled Monolayer) is formed in advance on the surface of a sensor chip (for example, a QCM sensor chip on which a gold thin film is formed), and the surface layer is formed. Alternatively, an ionic polymer may be applied or contacted on the side to form a bond between the functional groups of the two, and may be immobilized. In addition, a polymer film may be formed in situ using a radical living polymerization method in which an initiator is fixed directly on a substrate or with the SAM or silane coupling agent and the molecular length can be controlled by sequential reaction. Good.

-Surface charge density The sensor chip with a charged layer has a surface charge in order to place the ligand-carrying charged microparticles that may form a complex with the analyte at the desired density (preferably close packed). The density is preferably high enough to satisfy at least a certain level. The surface charge density of the sensor chip (charged layer) under the conditions in the step of bringing the sensor chip into contact with the charged fine particles, more specifically, the pH condition of the medium (aqueous solution) containing the ligand-carrying charged fine particles used in the process. Can be adjusted, for example, at 10 to 500 mC / m ² , preferably 50 to 200 mC / m ² . Such a surface charge density can be measured using a known means such as a zeta potential measurement device (for example, a zeta potential measurement system “ELSZ-2” (manufactured by Otsuka Electronics Co., Ltd.) using a flat sample cell). Can be measured. The surface charge density is preferably such that the number of ionic functional groups per projected cross-sectional area of the ligand-carrying charged fine particles is at least 1. By adjusting the conditions such as the number of ionic functional groups introduced per molecule of the ionic polymer used to form the ionic thin film and the amount of ionic polymer applied per unit volume of the sensor chip surface The surface charge density can be kept within a desired range.

-Surface roughness When the sensor chip is particularly for RIfS, it is ideal that the surface of the charged layer provided in the sensor chip is smooth. Such smoothness can typically be based on surface roughness. For example, the arithmetic average roughness (Rz) of the surface of the ionic functional group-modified sensor chip is preferably 1 to 50 nm, and more preferably 1 to 10 nm. Such surface roughness can be measured using a known means such as a surface roughness measuring device or an atomic force microscope (AFM).

-Ligand-supported charged fine particles-
In the present invention, in order to react with an analyte in a liquid phase to form a complex, a ligand-carrying charged fine particle composed of a charged fine particle and a ligand bound thereto is used.

-Charged fine particles As the charged fine particles, it is important to use positively or negatively charged fine particles having a size of usually about 1 to 100 nm for strong immobilization using electrostatic interaction. The size of such charged fine particles can be represented by a nominal value (catalog value) such as a volume average particle diameter measured by a dynamic light scattering method or a particle diameter of a product.

The dispersion form of the charged fine particles is preferably a self-emulsifying type having a strong bonding force, but can be used even if it is not a self-emulsifying type.
Various kinds of charged fine particles that can be used in the present invention are known, and are not particularly limited as long as they can support a ligand by an appropriate means as described later. Examples thereof include those composed of SiO ₂ and TiO ₂ , an ionic polymer, and a metal capable of fixing a ligand.

Charged fine particles made of SiO ₂ or TiO ₂ include so-called colloidal silica (for example, “Snowtech (registered trademark)” series, Nissan Chemical Industries, Ltd.), ultrafine particle titanium oxide (for example, “STT-65C-S”, titanium industrial stock). A company). These charged fine particles are positively charged when acidic and negatively charged when alkaline. TiO ₂ may be anatase type or rutile type.

  As the charged fine particles made of an ionic polymer, a substance known as a polymer colloid, a polymer latex or the like can be used. Such charged fine particles are positively or negatively charged in water depending on the ionic functional group of the ionic polymer. Examples of the ionic functional group include those similar to the cationic functional group and the anionic functional group described above regarding the charged layer. Moreover, as long as it aggregates in the form of particles, the same ionic polymer as described above with respect to the charged layer can be used as the charged fine particles.

  Examples of the metal may include nanoparticles such as gold, silver, copper, aluminum, and iron, or a composite having the metal oxide. Note that fine particles (typically colloidal particles) made of these metals are usually positively or negatively charged. For example, colloidal particles such as gold and silver are usually negatively charged. In order to immobilize the ligand on the surface of the charged fine particle, such a metal is a known method (for example, a method of treating with a predetermined compound and introducing a predetermined modifying group on the surface of the metal fine particle as described later). Anything that can be applied is acceptable. Moreover, metal fine particles (for example, antibody-immobilized gold nanoparticles) in which a ligand is immobilized in advance can be obtained as a product.

-Ligand A ligand is a substance that specifically binds to an analyte. For example, when the analyte is a protein, an antibody (including Fab, F (ab) _2, etc.) that recognizes and specifically binds a part thereof serves as a ligand, and when the analyte is a nucleic acid, it complements it. When a nucleic acid having a basic sequence is a ligand and the analyte is a molecule having a sugar chain, a lectin, which is a protein that recognizes and binds to the sugar chain, becomes a ligand. In addition, when the analyte is previously modified with a specific substance (for example, biotin), a substance (for example, avidin) that specifically binds to the specific substance may be used as a ligand.

  When the ligand is a protein (such as an antibody), there are modifying groups such as thiol group, amino group, carboxyl group, hydroxyl group at the terminal or side chain of the amino acid constituting the ligand. Can be used to combine with Nucleic acids, on the other hand, have amino groups and hydroxyl groups in their bases, but usually do not have carboxyl groups or thiol groups. Therefore, if necessary in relation to modifying groups, use known methods. These reactive groups may be introduced into the nucleic acid.

Production Method The ligand-carrying charged fine particles can be produced by performing a predetermined reaction for binding the charged fine particles and the ligand, usually in a liquid phase. The mode of binding between the charged fine particles and the ligand is not particularly limited, and a known method can be used.

For example, when the charged fine particles are composed of SiO ₂ , the charged fine particles are treated with a silane coupling agent to introduce a predetermined modifying group on the surface, while a reactive group corresponding to the modifying group is introduced. The ligand can be bound to the charged fine particles by preparing a ligand having the ligand and reacting these modifying groups and reactive groups. Similarly, when the charged fine particles are made of TiO ₂ , a silane coupling agent can be used.

  Even when the charged fine particles are made of a metal, an appropriate compound is selected and treated according to the metal, and a predetermined modifying group is introduced into the charged fine particles on the surface. What is necessary is just to make it react with the reactive group which has. For example, when the metal is gold, a SAM having a thiol group bonded to gold at one end and a modifying group such as a carboxyl group or an amino group at the other end can be used as the compound. In addition, charged fine particles into which a predetermined modifying group has been introduced in advance can be obtained as a product.

  In addition, when the charged fine particles are made of an ionic polymer, a part of an ionic functional group (for example, a carboxyl group which is an anionic functional group) of the ionic polymer is part of a reactive group (for example, amino group) of the ligand. The ligand can be bound to the charged fine particles by using the compound for reaction with the group.

  The amount of ligand carried on the charged fine particles can be set to a desired range by adjusting the number of modifying groups of the charged fine particles, the amount of ligand to be reacted (concentration of the ligand solution), and the like. It is appropriate that the molecular interaction with the charged layer is not disturbed.

  In addition, the reaction liquid at the time of producing the ligand-carrying charged fine particles is directly or diluted to an appropriate concentration, and mixed with an analyte (containing medium) in the subsequent complex formation step, and then the ligand-carrying charged fine particles -It can also be used to form an analyte complex. If the reaction liquid contains charged fine particles (unreacted charged fine particles) that are not bound to the ligand, they will remain in the complex formation process (without forming a complex with the analyte). In addition, since it is combined with the charged layer in the subsequent complex contact step, there is a possibility that the accuracy of the determination of the analyte is lowered. Therefore, the amount ratio of charged fine particles to be reacted and ligand is appropriately adjusted. Usually, the amount of the ligand is sufficiently increased with respect to the charged fine particles, so that unreacted charged fine particles are prevented from remaining as much as possible. desirable. Alternatively, for example, the ligand-carrying charged fine particles are purified by removing unreacted charged fine particles from the reaction solution by affinity chromatography, and the purified product is prepared with an aqueous solution having a predetermined concentration as necessary. It is desirable to use in this process.

-Intermolecular interaction measurement method-
The intermolecular interaction measurement method according to the present invention uses a sensor chip having a charged layer as described above, and contacts a separately formed ligand-carrying charged fine particle-analyte complex. Such intermolecular interaction measurement method is a procedure according to the conventional intermolecular interaction measurement method except for some steps, and using a measuring device similar to the conventional intermolecular interaction measurement method. Can be implemented.

(Embodiment of RIfS)
Hereinafter, an embodiment of a method for measuring an intermolecular interaction based on RIfS used in the embodiment of the present invention will be described with reference to FIG.

  The measurement system 1 for the intermolecular interaction measurement method mainly includes an intermolecular interaction measurement device 100 including a measurement member 10, a white light source 20, a spectroscope 30, a light transmission unit 40, a control device 50, and the like. It is composed of The white light source 20, the spectroscope 30, the light transmission unit 40, and the like are preferably housed in the main body of the measuring device 100, and the control device 50 in the form of, for example, a PC (Personal Computer) can be controlled in the main body of the measuring device 100. Connected to. Further, the measuring member 10 is generally rectangular and is preferably detachable from the measuring device 100 main body. The measuring member 10 (sensor chip 12 and flow cell 14) can be of a disposable type.

  The measurement member 10 includes a sensor chip 12 that is a place where optical interference to be measured is caused, and various media 60 (for example, ligand-carrying charged fine particles-analogue) that are usually mounted on the surface layer side of the sensor chip 12. And a flow cell 14 for forming a sealed channel 14b for feeding a sample solution including the light complex 62).

  In addition, as sensor chips for RIFS, etc., there are a wide variety of “channel type” structures in which a sample solution is fed with a pump or the like and brought into contact with the sensor chip 12 while flowing down the fine sealed channel 14b. As is known, it may be a “well type” structure in which various solutions are stored in a wider area, contacted with the sensor chip 12 and then rinsed.

  The sensor chip 12 basically includes a substrate 12a and an optical thin film 12b formed on the surface layer side thereof. In the present invention, the sensor chip 12 further includes a charged layer 12c formed on the surface layer side of the optical thin film 12b.

The substrate 12a is preferably made of, for example, Si (silicon). On the other hand, the optical thin film 12b is selected according to the material (refractive index) of the substrate so that the minimum reflectance wavelength observed when white light is used falls within an appropriate range (preferably in the visible light region). It is made of a material having a refractive index and a thickness. For example, when the substrate 12a is a Si substrate, the optical thin film 12b is an oxide film or nitride film such as SiN, Ta ₂ O ₅ , Nb ₂ O ₅ , HfO ₂ , ZrO ₂ , ITO (indium tin oxide). Is preferable, and a SiN film is particularly preferable. Each of these oxide films and nitride films has a refractive index of 1.8 to 2.4 in the visible light region (wavelength range of about 400 to 800 nm), and is an optical thin film formed on the surface layer side layer of the Si substrate. The optical thin film itself has the effect of suppressing non-specific adsorption to some extent while satisfying the performance. For example, by setting the thickness of the SiN film to about 45 to 90 nm, the bottom peak can be adjusted to a range of about 400 nm to 800 nm. The charged layer 12c is preferably formed of, for example, an ionic polymer.

  The flow cell 14 is a transparent member made of, for example, silicone rubber (polydimethylsiloxane: PDMS) that can be in close contact with the sensor chip 12. At least one 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 14 b is formed. The surface of the sensor chip located at the bottom of the sealed channel 14b becomes a reaction site 200 for observing a signal emitted by a predetermined intermolecular interaction. Both end portions of the groove 14a are exposed from the surface of the flow cell 14, one end portion is connected to a liquid feeding portion (for example, a syringe pump) and functions as an inlet 14c to which the medium 60 is supplied, and the other end portion Is connected to the waste liquid portion and functions as an outlet 14 d of the medium 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 the present 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 where a change in reflectance minimum 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 (reflection type RIfS). However, a light transmissive member is used as the measurement member 10 and the white light source 20 is used. It is also possible to arrange the spectroscope 30 so as to irradiate the measurement member 10 with the light and to receive the light transmitted through the measurement member 10 and to detect the spectral intensity of the transmitted light.

  The control device 50 receives the detection operation execution input from the operator and outputs a detection operation control execution command to the intermolecular interaction measurement 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 measuring light from the spectroscope 30, and calculates the reflectance by dividing the spectral intensity of the measuring light by the spectral intensity of the white light as a reference for each wavelength band. The spectral intensity data of the reference light may be previously measured and held 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 (λ) is determined. It is also possible to obtain the measured change amount (Δλ) of the minimum reflectance wavelength with respect to a certain minimum reflectance wavelength (baseline).

  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, with the control device 50, the white light source 20 is instructed in the intermolecular interaction measuring device 100 by an instruction from the control device 50. 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.

(Signal observation)
The intermolecular interaction measuring method according to the present invention comprises a step of contacting a ligand-carrying charged fine particle and an analyte in a medium (that is, in a homogeneous system) to form a ligand-carrying charged fine particle-analyte complex (hereinafter referred to as “composite”). A ligand-carrying charged fine particle-analyte complex is brought into contact with the charged layer formed on the ionic functional group-modified sensor chip as described above, and the intermolecular interaction between them. It includes a step of observing a signal that appears as a result of the action (hereinafter referred to as a “complex contact step”). Through these steps, a signal is observed and an analyte is detected.

-Analyte An analyte is a substance that specifically binds to a ligand. 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 that binds to a biomolecule such as a chemical substance or other biological substance can be an analyte. A tumor marker (for example, α-fetoprotein) present in blood, which is an important index in cancer diagnosis or the like, is an example of an important analyte. The analyte can also be modified in advance with a specific substance (for example, biotin). For such a modified analyte, a substance that specifically binds to the specific substance (for example, avidin) may be used as a ligand.

  In general, a specimen collected from a living body is used as a substance containing or possibly containing such an analyte. Examples of specimens include blood (serum / plasma) collected from humans and non-human animals (eg, mammals), urine, nasal fluid, saliva, feces, body cavity fluids (spinal fluid, ascites, pleural effusion, etc.) It is done. Such a specimen may be used after purification for removing impurities other than the analyte, if necessary.

-Complex formation step In order to form a ligand-carrying charged fine particle-analyte complex by reacting a ligand-carrying charged fine particle and an analyte in a medium, generally, a medium containing a ligand-carrying charged fine particle ( Hereinafter, a “ligand-supported charged fine particle solution”) and a medium containing an analyte (hereinafter referred to as “analyte solution”) may be mixed.

  As these “mediums”, it is preferable to use, for example, water which is widely used in the intermolecular interaction measurement method, that is, to prepare a ligand-carrying charged fine particle or an aqueous solution of an analyte. The water in this case may be an aqueous solution such as a buffer solution (for example, phosphate buffered saline) or other necessary reagents in addition to pure water. A medium capable of appropriately performing a predetermined measurement without interfering with the formation of a complex of a ligand-carrying charged fine particle and an analyte and the intermolecular interaction between the complex and the charged layer of the sensor chip performed thereafter. If so, a medium other than water may be used.

  The concentration and mixing amount of each of the ligand-supported charged fine particle solution and the analyte solution can be adjusted to a relatively appropriate range. Usually, a sufficient amount of charged ligand-bearing fine particles are present in the reaction solution so that at least all of the analyte forms a complex in order to detect or quantify all of the analyte contained in the analyte solution. Therefore, the concentration and mixing amount of the ligand-carrying charged fine particle solution and the analyte solution are adjusted.

  In preparing the ligand-carrying charged fine particle solution, a reaction solution for producing ligand-carrying charged fine particles can be used. It is also possible to adjust the concentration of the ligand-carrying charged fine particle solution used in the complex formation step by adjusting the amount of the ligand-carrying charged fine particle to be produced by adjusting the raw material solution for producing the ligand-carrying charged fine particle. .

  Further, since the concentration of the analyte solution is usually unknown, such as when the analyte in the specimen is actually measured, it is preferable to predict the concentration range of the analyte solution using a separate sample in advance.

Further, in the complex formation step, means such as stirring may be used in combination in order to promote the formation of the ligand-carrying charged fine particle-analyte complex.
Complex Contact Step Ligand-supported charged fine particle-analyte complex (hereinafter sometimes simply referred to as “complex”) is usually also referred to as a medium containing the complex (hereinafter referred to as “sample solution”). ) And is brought into contact with the charged layer through the medium.

  The “medium” containing the ligand-carrying charged fine particle-analyte complex may be the same as the “medium” containing the ligand-carrying charged fine particle or the “medium” containing the analyte.

  The sample solution may be a solution obtained by using the reaction solution in which the complex is formed in the above-described complex formation step as it is, or the reaction solution may be purified as necessary, or necessary reagents may be added. Or other processing. Such a sample solution usually contains both the ligand-carrying charged fine particle-analyte complex and the ligand-carrying charged fine particle itself (an unreacted substance that does not form a complex with the analyte). Both of these are captured by the charged layer and together form an optical thin film.

  Obtaining a predetermined measurement value and observing a signal (that is, change in measurement value) continuously before and after the complex contact step as described above, preferably before, during and after the start of the step. Thus, the ligand-supported charged fine particle-analyte complex (that is, the analyte itself) can be quantified. It is appropriate to determine that the process can be completed when the measured value no longer fluctuates after a certain period of time or when the fluctuation is expected to be sufficiently small.

For example, in the case of RIfS, the minimum value (λ) of the spectral reflectance of the reflected light is acquired as the measurement value. Furthermore, the change amount (Δλ ₁ ) of the measured value is calculated as the difference between the absolute value of the measured value (λ ₁ ) before the start of the complex contact process and the measured value (λ ₁ ′) after the end of the process. (Usually λ ₁ <λ ₁ ′ and Δλ ₁ = λ ₁ ′ −λ ₁ ). Such Δλ ₁ reflects the average film thickness of the optical thin film formed by both the ligand-carrying charged fine particle-analyte complex trapped in the charged layer and the unreacted ligand-carrying charged fine particle. .

The amount of analyte contained in a sample solution can be quantitatively evaluated by comparing Δλ ₁ for the sample solution with a reference value obtained using a sample having a known concentration. A relative evaluation can also be made by comparing with Δλ ₁ for the sample solution.

For example, in the same procedure as the complex formation step described above, by using a reaction liquid obtained by adding at least two predetermined reference amounts (concentrations) of analyte to the ligand-carrying charged fine particles, The relationship between the amount of the analyte and the measured value (or the change amount of the measured value), more specifically, the relational expression can be acquired. At this time, it is preferable to prepare a reaction solution obtained by setting one of the reference amounts to zero, that is, without adding any analyte. When the reference amount of the analyte is zero, a test liquid containing only the ligand-carrying charged fine particles without containing the ligand-carrying charged fine particle-analyte complex is obtained. When this test solution is brought into contact with the sensor chip, an optical thin film consisting only of ligand-carrying charged fine particles is formed on the surface layer of the charged layer. On the other hand, if the reference amount of the analyte is other than zero, depending on the reference amount, some of the ligand-carrying charged fine particles form a complex with the analyte, and the remaining ligand-carrying charged fine particles remain unreacted. The test solution is obtained. When this test solution is brought into contact with the sensor chip, an optical thin film composed of both the ligand-carrying charged fine particle-analyte complex and the ligand-carrying charged fine particle is formed on the surface layer of the charged layer. At this time, since there is a difference between the height of the former and the latter, there is a difference in the overall thickness of the optical thin film to be formed, that is, the measured λ ₁ depending on the proportion of the former and the latter. become. If the reference amount of the analyte is excessive with respect to the ligand-carrying charged fine particles, substantially no unreacted ligand-carrying charged fine particles are contained, and only the ligand-carrying charged fine particle-analyte complex is contained. A test solution is obtained. Based on the relational expression that can be obtained from such a test solution, the amount (concentration) of the analyte in the sample solution can be estimated from the measured value (or the amount of change in the measured value) in the sample solution.

  In addition, the analyte can be similarly detected by using the measurement value in the QCM instead of the measurement value in the RIfS as described above. In QCM, the resonance frequency is measured, and when a ligand-carrying charged fine particle-analyte complex and / or unreacted ligand-carrying charged fine particle is captured on the surface of the sensor chip, the change can be observed.

  In the following examples, “MI-Affinity” (registered trademark, Konica Minolta Opto Co., Ltd.) is used as a RifS-type intermolecular interaction measuring apparatus, and the above-mentioned “MI- Affinity dedicated sensor chip (substrate: silicon wafer, optical thin film: silicon nitride) and flow cell (PDMS, width 2.5 mm x length 16 mm x depth 0.1 mm, and 1 mm in diameter at both ends of the groove) As the syringe pump, “Econoflo Syringe Pump 70-2205” (manufactured by Harvard Apparatus) was used.

[Preparation Example 1] Cationic Modified Sensor Chip Cationic aqueous polyurethane resin “Hydran CP-7610” (manufactured by DIC Corporation, volume average particle size 8 nm) was diluted with water to prepare a coating solution having a concentration of 1 wt%. This coating solution was applied to the surface of an unmodified sensor chip using a spin coater (rotation number: 3000 rpm). After the application, the film was dried at 70 ° C. for 30 minutes to produce a cationic modified sensor chip on which a film made of the cationic polyurethane was formed. An atomic force microscope (AFM) image of the surface of the cationic modified sensor chip is shown in FIG.

[Preparation Example 2-1] Ligand-supported charged fine particles (SiO ₂ )
As the SiO ₂ fine particles, “Snowtex XS” (manufactured by Nissan Chemical Industries, Ltd., volume average particle diameter 5 nm) was used. Further, an anti-α-fetoprotein [AFP] mouse monoclonal antibody (clone: 1D5; Mikuri Immuno Laboratory Co., Ltd.) was used as a ligand. 10 mL of a 5% solution of “Snowtex XS” was collected, 0.5 mL of 5-carboxy-pentyltriethoxysilane was added, and reacted at room temperature for 3 hours. Furthermore, 1 mL of 25 mM MES (2-morpholinoethanesulfonic acid) buffer (pH 5.5), 100 equivalents of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Dojindo Laboratories) ) And N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) were added to activate the carboxyl group. 1 mL of AFP antibody 1D5 was added and reacted at room temperature for 30 minutes to react 1D5 with microparticles. Unreacted antibody was removed using a spin column.

[Preparation Example 2-2] Ligand-supported charged fine particles (anionic polymer)
As the anionic polymer fine particles, “Hydran WLS 202” (manufactured by DIC Corporation) was used. Further, an anti-α-fetoprotein [AFP] mouse monoclonal antibody (clone: 1D5; Mikuri Immuno Laboratory Co., Ltd.) was used as a ligand. 20 mL of a 10% solution of “Hydran WLS 202” was collected, 1.5 mL of 25 mM MES (2-morpholinoethanesulfonic acid) buffer (pH 5.5), 100 equivalents of 1-ethyl-3- (3-dimethylaminopropyl) Carbodiimide hydrochloride (EDC: manufactured by Dojindo Laboratories) and N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) were added to activate the carboxyl group. 1 mL of 1 mg / mL AFP antibody 1D5 was added to the above solution and reacted at room temperature for 30 minutes to react 1D5 with microparticles. Unreacted antibody was removed using a spin column.

[Example 1]
(Measurement preparation process)
The RifS intermolecular interaction measurement apparatus “MI-Affinity” was turned on and waited for about 20 minutes until the light source was stabilized.

  A flow cell was loaded on the ionic (cationic) film sensor chip produced in Production Example 1 to construct a measurement member in which a sealed channel was formed. The measurement member was set in the measurement device, and a syringe pump was used to send the liquid from the outside of the measurement device to the sealed flow path so as to be brought into contact with the sensor chip surface.

(Composite formation process)
1 μg / mL, 10 μg / ml of an aqueous solution containing the ligand-carrying charged fine particles (SiO ₂ ) prepared in Preparation Example 2-1 at a concentration of 1% by weight, and α-fetoprotein (AFP) (manufactured by Cris Antibodies GmbH) as an analyte, A PBS solution (pH 7.4; manufactured by Nacalai Tesque) containing 100 μg / ml was mixed to form a ligand-supported charged fine particle-analyte complex.

(Composite contact process)
The reaction liquid obtained by the complex formation step is introduced into the sealed flow path of the measurement member for 10 minutes (total 200 μL) at a liquid feed speed of 20 μL / min by the syringe pump, and a sensor chip on which a charged layer is formed The surface was contacted. The measured values are shown in Table 1. From the results in the table below, it can be seen that the AFP assay is quantitatively and highly sensitive.

[Example 2]
In the complex formation step, in place of the aqueous solution containing the ligand-carrying charged fine particles (SiO ₂ ) produced in Production Example 2-1, the ligand-carrying charged fine particles (anionic polymer) produced in Production Example 2-2 are contained. The measurement was performed in the same procedure as in Example 1 except that the aqueous solution used was used. The measured values are shown in Table 2. From the results in the table below, it can be seen that the AFP assay is quantitatively and highly sensitive.

DESCRIPTION OF SYMBOLS 1 Measurement system 10 Measuring member 12 Sensor chip 12a Substrate 12b Optical thin film 12c Charged layer 14 Flow cell 14a Groove 14b Sealed flow path 14c Inlet 14d Outlet 20 White light source 30 Spectroscope 40 Light transmission part 41 First optical fiber 42 Second Optical fiber 50 Control device 60 Medium 62 Ligand-supported charged fine particle-analyte complex 100 Intermolecular interaction measurement device 200 Measurement site

Claims (8)

  A ligand-carrying charged fine particle comprising a charged fine particle charged positively or negatively and a ligand bound thereto. _2. The ligand-carrying charged fine particles according to claim 1, wherein the charged fine particles are made of SiO ₂ , TiO ₂ , an ionic polymer, or a metal capable of fixing a ligand. An intermolecular interaction measurement method using a sensor chip having a charged layer that is positively or negatively charged on a surface layer,
The step of reacting the ligand-carrying charged fine particles according to claim 1 or 2 charged in the opposite direction to the charged layer and an analyte in a medium to form a ligand-carrying charged fine particle-analyte complex, and A method for measuring an intermolecular interaction comprising a step of contacting a medium containing a ligand-carrying charged fine particle-analyte complex with the surface of the charged layer and observing a signal appearing when the intermolecular interaction works. .   The intermolecular interaction measurement method according to claim 3, wherein the charged layer is a layer made of an ionic polymer.   The intermolecular interaction measurement method according to claim 3 or 4, wherein the ionic polymer is a polymer having a cationic functional group. The surface charge density of the charged layer is in the range of 50~200mC / m ^2, intermolecular interaction measurement method according to any one of claims 3-5.   The method for measuring an intermolecular interaction according to any one of claims 3 to 6, wherein the arithmetic average roughness of the surface of the charged layer is in the range of 1 to 50 nm.   The sensor chip according to claim 3, wherein the sensor chip is for RIfS or QCM.