JP2013029370A - Local-field optical sensor chip having ligand highly densely disposed thereon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor chip for an analyte detection method using surface plasmon resonance generated on a metal thin film, the sensor chip on which ligand is immobilized highly densely more than a conventional one.SOLUTION: A ligand-immobilized local-field optical sensor chip for detecting analytes includes: at least a dielectric member; a metal thin film formed on the dielectric member; a positively or negatively charged layer formed on the metal thin film; and an immobilized ligand layer formed of ligand-carrying charged particles comprising charged particles that are formed on the charged layer, immobilized by electrostatic interaction, and charged inversely to the charged layer, and ligand bonded thereto.

Description

  The present invention relates to a sensor chip (local field light sensor chip) for an analyte detection method using localized field light (evanescent wave) represented by SPFS and SPR, and an analyte using the sensor chip. It relates to the detection method.

  In recent years, analytes (substances to be detected) such as biomolecules and organic polymers can be detected on the surface of a sensor chip (typically a measurement member manufactured in a chip shape) between the analyte and the analyte. Research and development of a method for detecting analytes directly and quantitatively by capturing using a ligand (capture substance) with an intermolecular interaction and observing the specific signal emitted by the ligand is underway. . 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.

  For example, the following SPR (Surface Plasmon Resonance) is known as a method for detecting an analyte without labeling, that is, without using a label such as a radioactive substance or a phosphor. When the measurement light is irradiated at a predetermined incident angle at which total reflection occurs on the metal thin film formed on the dielectric member, the intensity of the reflected light is attenuated due to surface plasmon resonance in the metal thin film (total reflection attenuation: ATR). The intensity of the reflected light is minimal at the incident angle. Here, when the analyte is captured on the sensor (metal thin film), the dielectric constant changes, and the incident angle at which the intensity of the reflected light is minimized changes. In SPR, an analyte can be detected by measuring the incident angle as a signal.

  On the other hand, as a method for detecting an analyte using a phosphor, for example, a metal thin film formed on a dielectric member is irradiated with measurement light (excitation light) at an angle at which total reflection attenuation (ATR) occurs, and a metal The evanescent wave (non-propagating light, localized field light) transmitted through the thin film is trapped in the vicinity of the metal thin film by utilizing the fact that it is enhanced several tens to several hundreds of times by resonance with the surface plasmon of the metal thin film. Also known is SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy), which detects the analyte by efficiently exciting the phosphor labeled with the analyte and measuring the fluorescence signal. It has been.

  Now, immobilization of a ligand on a conventional sensor chip has been performed by the following method. That is, a modifying group capable of causing a specific bond is introduced on the surface of the sensor chip, while a reactive group corresponding to the modifying group is introduced into the ligand, and these modifying group and reactive group are reacted. By binding, the ligand was immobilized on the sensor chip.

  For example, in the case of a sensor chip for SPFS, a metal thin film (for example, Au) is first formed on a dielectric member, and in many cases, in order to avoid metal quenching caused by contact between the phosphor and the metal thin film. Then, another dielectric layer is formed on the surface of the metal thin film to produce an unmodified sensor chip. The surface of the dielectric layer of such an unmodified sensor chip is treated with, for example, a silane coupling agent having an amino group at the terminal to be modified with an amino group, followed by NHS (N-hydroxysuccinimide) -PEG4. -Sensor chip in which the ligand is immobilized on the surface by treating with biotin, binding biotin to the amino group, reacting this biotin with avidin, and then reacting with a biotinylated ligand (for example, antibody) Can be produced. Further, the unmodified sensor chip is treated with a silane coupling agent having a carboxyl group at the end to be modified with a carboxyl group, followed by EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) and NHS A sensor chip in which the ligand is immobilized on the surface is also produced by reacting with an active ester of the carboxyl group and then reacting with a ligand having an amino group (typically a protein such as an antibody). be able to.

  On the other hand, it is known that charged polymer particles form a single particle film on a charged solid surface having an opposite charge by electrostatic interaction. For example, Non-Patent Document 1 discloses a cation that can be efficiently synthesized by soap-free emulsion copolymerization of styrene (ST) and a water-soluble methacrylic acid ester and is derived from a relatively high concentration of sulfonium groups on the surface. When a glass substrate that exhibits a negative surface charge in water is immersed in a charged latex of P (ST-co-MAPDS) fine particles, adsorption due to electrostatic interaction occurs and is desorbed even by ultrasonic irradiation. It is described that a relatively stable single particle film can be obtained. However, the method described in the above document is derived from electrostatic charging on the glass surface, and cannot maintain stability over time. The charge used for the electrostatic interaction known in the above-mentioned document and other documents is static, or because it uses an electric field, it deteriorates with time, and the stability of the single particle layer cannot be ensured, so that it is difficult to develop the sensor. . The fine particles described in the above literature are relatively coarse particles of around 230 nm, and therefore use the advection and accumulation principle and sedimentation, and are unlikely to be pure electrostatic interactions. Further, there is no known document that mentions that the amount of immobilized ligand, the amount of effective antibody, etc. can be increased by using electrostatic interaction.

  Patent Document 1 discloses a surface of a glass substrate by immersing a glass substrate in a methanol solution of 3-aminopropyltrimethoxysilane and then a gold colloid solution for a localized plasmon resonance sensor. A method of forming a gold colloid monolayer film, and when a predetermined receptor is adsorbed to the gold colloid, the adsorption of the substance is detected from a change in absorbance when the predetermined substance is adsorbed to the receptor. The use as an affinity sensor is described.

Japanese Unexamined Patent Publication No. 2000-356587 (Japanese Patent No. 3452837)

Masaru Nagai, Keizo Yamaguchi "Single-particle film formation of polymer colloids on solid substrates" Journal of the Textile Society of Japan, Vol. 60, No. 4, pp.P-90-P-95 (2004).

When the conventional method using the silane coupling agent as described above is employed, the ligand immobilized on the surface of the sensor chip has problems such as low density, unevenness, and uneven alignment. In particular, in the case of a sensor chip for SPFS, providing a dielectric layer made of SiO ₂ or the like on the surface is suitable and frequently used to avoid metal quenching, but an amino group or a carboxyl group is introduced on the surface. The silane coupling agent (silanol group or alkoxysilyl group present at one end thereof) used for this purpose is introduced through a bond with a silanol group produced by the reaction of SiO ₂ with water in the air. However, since there are few silanol groups on the sensor chip side generated in this way, the method using a silane coupling agent has the density of amino groups and carboxyl groups that can be introduced, that is, the density of the ligand finally introduced. There was a limit. It is also known that in the reaction of an amino group or carboxyl group with a ligand, the amount of immobilized ligand can be increased by controlling the pH, but the number of effective antibodies is rather decreased because the orientation is impaired.

  It is an object of the present invention to provide a sensor chip for an analyte detection method using surface plasmon resonance that occurs in a metal thin film, in which a ligand is immobilized at a higher density that is more effective than conventional ones.

  The inventors first introduce high-density ionic functional groups having a positive or negative charge, such as by applying an ionic polymer to the surface of a localized field photosensor chip (for SPFS, etc.). I found out that I can do it. In addition, the ligand is supported on ionic fine particles (charged fine particles) having a charge opposite to that of the ionic functional group, and these are bonded to the ionic functional group by electrostatic interaction, so that the ligand is homogeneous. It has been found that it can be immobilized on the surface of the sensor chip at a high density, and that the signal can be observed with higher sensitivity and higher accuracy than before by using the sensor chip with the ligand immobilized in this way. The present invention has been completed.

That is, the present invention includes the following matters.
[1] A ligand-immobilized localized field optical sensor chip for analyte detection, comprising at least
A dielectric member;
A metal thin film formed on the dielectric member;
A positively or negatively charged layer formed on the metal thin film;
An immobilized ligand layer composed of a ligand-carrying charged fine particle that is formed on the charged layer and is immobilized by electrostatic interaction and is composed of charged fine particles opposite to the charged layer and a ligand bonded thereto. And a ligand-immobilized localized field photosensor chip.

[2] The sensor chip according to [1], wherein the charged layer is a layer made of an ionic polymer.
[3] The sensor chip according to [1] or [2], wherein the ionic polymer is a polymer having a cationic functional group.

[4] The sensor chip according to any one of [1] to [3], wherein a surface charge density of the charged layer is in a range of 50 to 200 mC / m ² .
[5] The sensor chip according to any one of [1] to [4], wherein an arithmetic average roughness of a surface of the charged layer is in a range of 1 to 50 nm.

  [6] [1] to [5], wherein the sensor chip is for SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) or SPR (Surface Plasmon Resonance). The sensor chip according to any one of the above.

[7] wherein the charged particles, SiO _2, TiO _2, is made of an ionic polymer, or the ligand from lockable metal sensor chip according to any one of claims 1 to 6.

[8] A method for producing a ligand-immobilized localized field photosensor chip for analyte detection according to any one of [1] to [7],
Charged positively or negatively charged charged layer provided in the local field photosensor chip is contacted with charged fine particles oppositely charged to the charged layer and ligand-carrying charged fine particles composed of ligands bound to the charged fine particles. A production method comprising a step of forming an immobilized ligand layer comprising the ligand-carrying charged fine particles by immobilization by interaction.

[9] An analyte detection method using the ligand-immobilized localized field photosensor chip according to any one of [1] to [7], comprising at least
A step of bringing an analyte-containing medium into contact with an immobilized ligand layer in a measurement region of the ligand-immobilized localized field optical sensor chip; and a surface plasmon that occurs in the metal thin film by irradiating the measurement region with the measurement light An analyte detection method comprising a step of observing a signal based on resonance.

  According to the present invention, it is possible to immobilize charged fine particles having a reverse charge and carrying a ligand at a high density (in a close-packed state) by a charge of an ionic functional group provided on the surface of the sensor chip. it can. As a result, the ligands can be arranged uniformly and at a high density, and the orientation of the ligands can be made uniform. Therefore, by using such a sensor chip according to the present invention, it is possible to detect and quantify analytes with higher sensitivity and higher accuracy than before.

FIG. 1 is a schematic diagram of an example of an embodiment of the present invention. FIG. 2 is an atomic force microscope (AFM) image of the surface of the sensor chip after the charged layer formation step of a sample manufactured by the same method as in Example 1. FIG. 3 is an atomic force microscope (AFM) image of the surface of the sensor chip after the immobilized ligand layer forming step of the sample produced by the same method as in Example 1. FIG. 4 shows the results of Example 1, in which the charged layer formation process ends (“cation” in the graph), the immobilized ligand layer formation process ends (“cation + Ab” in the graph), and the analyte contact process ends (“ It is a graph showing the reflection intensity corresponding to the signal in SPR method measured by each of cation + Ab + Ag "). FIG. 5 shows, in Example 1, the end point of the charged layer formation step (“cation” in the graph), the end point of the immobilized ligand layer formation step (“cation + Ab” in the graph), the end point of the analyte contact step (“ It is a graph showing the fluorescence intensity corresponding to the signal in SPFS method measured by cation + Ab + Ag ”). 6 is an atomic force microscope (AFM) image of the surface of the sensor chip after the analyte contact step in Example 1. FIG.

  In the present invention, an analyte detection method using a “localized field light sensor chip” is a measurement member (sensor chip) that generates a localized field light (evanescent wave) and is immobilized on the surface thereof. It is a general term for methods for detecting or quantitatively measuring an analyte by observing a signal based on specific binding between a predetermined molecule (ligand) and a molecule to be detected (analyte). Such analyte detection methods typically include SPFS and SPR, and also include measurement methods using evanescent (total reflection) and evanescent fluorescence (total reflection fluorescence). In the present specification, the “localized field light sensor chip” may be simply referred to as a “sensor chip”.

All of these analyte measuring methods are known, and the outline thereof is as follows.
SPR (Surface Plasmon Resonance) is the following method. When measuring light is irradiated onto the metal thin film formed on the dielectric member while scanning the angle, the intensity of the reflected light is attenuated by surface plasmon resonance in the metal thin film (total reflection attenuation: ATR), and the reflected light is reflected at a certain incident angle. The strength of is minimal. Here, when the analyte is captured on the sensor (metal thin film), the dielectric constant changes, and the incident angle at which the intensity of the reflected light is minimized changes. In SPR, an analyte can be detected by measuring the incident angle as a signal.

  SPFS (Surface Plasmon-field Enhanced Fluorescence Spectroscopy) irradiates a metal thin film with an excitation light at an angle where total reflection attenuation (ATR) occurs on a metal thin film formed on a dielectric member. Using the fact that the transmitted evanescent wave (localized field light) is enhanced several tens to several hundreds of times by resonance with the surface plasmon of the metal thin film, the analyte captured in the vicinity of the metal thin film is labeled. This is a method for efficiently exciting a fluorescent substance and measuring the fluorescence signal.

  For evanescent (total reflection), the sample is brought into close contact with a high refractive index medium (prism) that is transparent from the visible light to the infrared region, and the minimum value is obtained in the same way as SPR by total reflection attenuation (ATR) measurement. It can be measured by quantity. Unlike SPR, since no metal is used, the electric field enhancement is not large, but the sensor performance is highly stable.

  Evanescent fluorescence (total reflection fluorescence) irradiates excitation light at an angle where total reflection attenuation (ATR) occurs on a metal thin film formed on a dielectric member, and evanescent waves (localized field light) transmitted through the metal thin film. This is a method of efficiently exciting a phosphor labeled with an analyte trapped in the vicinity of a metal thin film and measuring the fluorescence signal by using the fact that it is enhanced several times to ten times.

  Hereinafter, the present invention will be described in more detail with a focus on SPFS and SPR embodiments. However, the analyte detection method in the present invention is not limited to SPFS and SPR. A person skilled in the art can develop the present invention in other analyte detection methods using surface plasmon resonance occurring in a metal thin film, based on the description of the present specification.

  In the following description of the sensor chip, for convenience, the side on which the ligand is finally immobilized is referred to as “upper” or “front”, and the opposite side is referred to as “lower” or “back”. In addition, “to form on” a certain substance is not limited to the case where it is formed directly on (in direct contact with) that substance, but within the range that does not hinder the practice of the invention. It is also included in the case of forming (without direct contact).

-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, an immobilized ligand layer made of ligand-carrying charged fine particles is further formed on the surface of this ionic functional group-modified sensor chip to produce a ligand-immobilized sensor chip, and then an analyte (including medium) is brought into contact therewith. And used for measurement to detect the analyte.

(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 analyte detection method. An unmodified sensor chip for SPFS and SPR (hereinafter also referred to as “sensor base material”) generally has at least a substrate made of a dielectric member (for example, SiO ₂ ) and a surface layer side thereof. A formed metal thin film (for example, Au). The sensor substrate for total reflection and total reflection fluorescence is generally composed of a dielectric member (for example, a high refractive index glass or a high refractive index resin chip).

  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 such a coating method is used, a coating solution is prepared by dissolving in an appropriate solvent in an appropriate solvent (for example, 0.1 to 1% by weight) according to the type of ionic polymer. May be applied. Generally, it is possible to form a charged layer made of an ionic polymer by adjusting the concentration of the ionic polymer in the coating solution to 0.5 to 10% by weight and the coating film thickness in the range of 5 to 100 nm. it can.

  If necessary, a SAM (Self-Assembled Monolayer) is formed in advance on the surface of a sensor chip (for example, a sensor chip for SPR or SPFS on which a gold thin film is formed) An embodiment may be adopted in which an ionic polymer is applied or brought into contact with the surface layer side to form a bond between both functional groups to 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 A sensor chip with a charged layer has a surface charge density that is high enough to satisfy at least a certain level in order to arrange ligand-carrying charged fine particles at a desired density (preferably in close packing). preferable. 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 Ideally, the surface of the charged layer provided in the sensor chip should be smooth in order to increase measurement accuracy. 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).

(Configuration of sensor substrate)
An unmodified sensor chip (sensor base material) for SPFS and SPR includes at least a dielectric member (prism or transparent flat substrate), a metal thin film formed on the dielectric member, and, if necessary, a spacer. It is comprised by a layer, A well-known general thing can be used also in this invention.

-Dielectric member The dielectric member which comprises a sensor base material may be prism shape, or a planar substrate shape. In the former case, a metal thin film or the like is formed on the horizontal surface of the dielectric member, and the obtained sensor chip is integrated with a prism that irradiates measurement light. In the latter case, the metal thin film or the like is formed on a plane on one side of the dielectric member, and the obtained sensor chip is used in close contact with the horizontal plane of the prism that irradiates the measurement light, separated from the prism. Will be.

The dielectric member, glass, polycarbonate (PC), a plastic such as cycloolefin polymer (COP), ranges preferably the d-line refractive index (588 nm) [n _d] is 1.40 to 2.20 Substances in the above can be used. When the dielectric member is manufactured as a transparent flat substrate, the thickness can be adjusted within a range of 0.01 to 10 mm, for example. Before forming the metal thin film, the surface of the dielectric member is preferably subjected to a cleaning treatment with acid or plasma.

Metal thin film The metal thin film constituting the sensor substrate is at least one selected from the group consisting of gold, silver, aluminum, copper, and platinum that is stable against oxidation and has a large electric field enhancement effect by surface plasmons. It is preferable that it consists of these metals (it may be the form of an alloy), and it is especially preferable that it consists of gold.

  Examples of the method for forming the metal thin film on the dielectric member include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. Since it is easy to adjust the thin film formation conditions, it is preferable to form a chromium thin film and a metal thin film by sputtering or vapor deposition.

  When a glass dielectric member is used, it is preferable to previously form a thin film of chromium, nickel chromium alloy or titanium in order to more firmly bond the glass and the metal thin film.

  The thickness of the metal thin film made of gold, silver, aluminum, copper, platinum, or an alloy thereof is preferably 5 to 500 nm, and the thickness of the chromium thin film is preferably 1 to 20 nm so that surface plasmons are easily generated. From the viewpoint of the electric field enhancement effect, gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm are more preferable, The thickness of the chromium thin film is more preferably 1 to 3 nm.

(Spacer layer)
If necessary, a spacer layer made of a dielectric material may be formed on the sensor base material on the metal thin film in order to prevent metal quenching of the fluorescent dye by the metal thin film. In this case, the charged layer is formed on the spacer layer, that is, formed on the metal thin film with the spacer layer interposed therebetween.

As the dielectric, various optically transparent inorganic substances and natural or synthetic polymers can be used. Among these, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferably used because of excellent chemical stability, production stability, and optical transparency.

  The thickness of the spacer layer is usually 10 nm to 1 mm, and preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, the thickness is preferably 200 nm to 1 mm from the viewpoint of the electric field enhancement effect, and more preferably 400 nm to 1,600 nm from the stability of the electric field enhancement effect.

  Such a spacer layer can be formed by a known means such as a sputtering method, an electron beam evaporation method, a thermal evaporation method, a method using a chemical reaction using a material such as polysilazane, or a spin coater.

-Ligand-supported charged fine particles-
In the present invention, in order to form an immobilized ligand layer further on the surface layer side of the charged layer, ligand-carrying charged fine particles composed of charged fine particles and a ligand bonded thereto are 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 The ligand is not particularly limited as long as it is a substance that specifically binds to the 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.

  The reaction liquid when preparing the ligand-carrying charged fine particles is left as it is or diluted to an appropriate concentration, and in the subsequent immobilized ligand layer forming step, it is brought into contact with a sensor chip equipped with a charged layer to charge the ligand-carrying charge. It can also be used to form a fine particle layer. If charged fine particles not bound to the ligand (unreacted charged fine particles) are contained in the reaction solution, they are also bonded to the charged layer, and accordingly, the amount of ligand-carrying charged fine particles bound to the charged layer. (Density) may decrease. 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.

-Ligand-immobilized sensor chip In the method for detecting an analyte in the present invention, an immobilized ligand is used according to the method (step) described below, using an ionic functional group-modified sensor chip and a ligand-carrying charged fine particle as described above. It can be produced by forming a layer.

(Immobilized ligand layer formation process)
A sensor chip having an immobilized ligand layer (ligand-immobilized sensor chip) is loaded with a ligand on a charged layer that has been formed on the surface of the sensor chip in advance by applying, for example, an ionic polymer. It can be produced by a production method including a step of forming an immobilized ligand layer composed of ligand-carrying charged fine particles (immobilized ligand layer forming step) by bringing charged fine particles into contact with each other and immobilizing them by electrostatic interaction.

  Usually, the contact of the ligand-carrying charged fine particles with the charged layer is preferably performed by bringing a medium containing the ligand-carrying charged fine particles into contact with the charged layer. As the medium, for example, water that is widely used in the analyte detection method, that is, an aqueous solution of ligand-carrying charged fine particles is preferably used. 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 other than water may be used as long as the ligand-carrying charged fine particles can be immobilized on the charged layer by electrostatic interaction.

  Further, the method of contacting the charged layer of the medium containing the ligand-carrying charged fine particles is not particularly limited, and the medium may be brought into contact (moving down) or in a stationary state. You may make it contact. As the former contact mode, a flow path type measurement member is constructed, and a medium containing ligand-carrying charged fine particles is introduced from a closed flow path formed by a flow cell into a sensor chip in which a charged layer is formed in advance. By doing so, it is preferable to contact the surface of the sensor chip. Such an embodiment requires a shorter time until the coating with the ligand-carrying charged fine particles reaches a sufficient level as compared with the latter embodiment described below. As the latter contact mode, a sensor chip in which a charged layer is formed in advance may be simply immersed in a medium containing ligand-carrying charged fine particles contained in a container, or a charged layer is formed. It is also possible to construct a well-type measurement member whose bottom surface is a sensor chip, and inject a medium containing ligand-carrying charged fine particles into the well. In any case, the sensor chip may be washed as necessary after contacting the medium containing the ligand-carrying charged fine particles. In the case of contact in a well-type structure, shaking, vortexing, stirring, ultrasonic waves, or the like may be used as necessary to increase efficiency.

  Various conditions such as the concentration of the ligand-carrying charged fine particles in the medium and the contact time between the charged layer and the medium (liquid feeding speed and liquid feeding time of the medium) are not particularly limited, Conditions under which an immobilized ligand layer is formed may be employed. For example, in order to ensure that the surface of the sensor chip is sufficiently (closely packed) with the ligand-carrying charged fine particles, the concentration of the ligand-carrying charged fine particles in the medium is sufficiently high and / or charged. It is preferable that the contact time between the layer and the medium is sufficiently long. On the other hand, it is also possible to adjust the density of the ligand immobilized on the sensor chip by adjusting the concentration of the ligand-carrying charged fine particles in the medium. The concentration of the ligand-carrying charged fine particles in the medium can be adjusted, for example, in the range of 0.01 to 1 wt%.

  As described above, when the immobilized ligand layer forming step is performed in such a manner that the medium containing the ligand-carrying charged fine particles is fed into the sealed channel and brought into contact with the sensor chip, an appropriate processing time is determined. Therefore, the measurement of the signal may be started before the start of the processing step and continuously measured in real time thereafter. In particular, in SPR, through observation of how the measured value changes with the lapse of processing time (the incident angle at which the reflection intensity is minimized), the ligand-carrying charged fine particles are bound to the charged layer, and the immobilized ligand layer is The state of formation can be confirmed at any time. For example, when the measured value does not change or the fluctuation is sufficiently small, it is determined that the binding of the ligand-carrying charged fine particles, that is, the formation of the immobilized ligand layer has reached saturation (close-packing), and the immobilization is performed. The ligand layer forming step can be terminated. It is also possible to determine that the formation of the immobilized ligand layer has reached the desired level when the change in the measured value reaches a predetermined level, and terminate the immobilized ligand layer formation process at that stage. is there.

-Analyte detection method-
The analyte detection method according to the present invention uses a sensor chip including a charged layer and an immobilized ligand layer formed on the surface side as described above.

  Such an analyte detection method is basically the same procedure as in the case of using a sensor chip on which a ligand is immobilized by a conventional method (for example, using a silane coupling agent), and the same measurement apparatus. Can be implemented.

-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.

-Measuring member construction method The measuring member construction method is common to SPFS and SPR.
In the case of a “channel type” system in which various media (for example, a sample solution containing an analyte) are fed through a closed channel and brought into contact with a measurement region, the sensor chip as described above (in the present invention, a ligand) A measurement member is constructed by loading and fixing a “flow cell” which is a member for forming a sealed channel on the surface layer side of the immobilized sensor chip).

  On the other hand, in the case of a “well type” system in which various media are brought into contact with the measurement area in a wider area, a through hole is provided on the surface layer side of the sensor chip as described above (in the present invention, a ligand-immobilized sensor chip). The measurement member is constructed by loading and fixing the “well member” having it.

  When fixing the flow cell or well member on the sensor chip, it is preferable to use an adhesive, matching oil, transparent adhesive sheet or the like having the same optical refractive index as that of the dielectric member.

  Hereinafter, an analyte detection method will be described in accordance with embodiments of SPFS and SPR, which are typical analyte detection methods using surface plasmon resonance that occurs in a metal thin film.

<SPR Embodiment>
The measurement system for SPR is mainly provided on the measurement member, the dielectric member side of the measurement member, a light source for irradiating measurement light (incident light) from the back side of the metal thin film in the measurement region, and the measurement light And a light receiver for receiving the reflected light.

In the case of conforming to SPR, specifically, the analyte can be detected by performing the following steps using the system as described above.
(1) Analyte contact step: a step of bringing a medium containing an analyte into contact with a measurement region where the immobilized ligand layer of the ligand-immobilized sensor chip as described above is formed;
(2) Signal observation step; a step of irradiating measurement light to the measurement region that has undergone the above steps and observing a signal based on surface plasmon resonance that occurs in the metal thin film.

(Analyte contact process)
For the analyte, a medium containing the analyte (hereinafter sometimes referred to as “sample solution”) is prepared and brought into contact with the immobilized ligand layer via the medium.

  As the medium containing the analyte, for example, it is preferable to use water which is widely used in the intermolecular interaction measurement method, that is, to prepare an aqueous solution of the 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 other than water can be used as long as the medium can appropriately perform a predetermined measurement without preventing specific binding between the immobilized ligand and the analyte.

(Signal observation process)
In the case of SPR, a signal based on surface plasmon resonance that occurs in a metal thin film reflects the intensity of reflected light attenuated by surface plasmon resonance. That is, the incident angle (θ) of the incident light when the intensity of the reflected light is minimized is acquired as a measurement value. In general, the amount of change in the measured value (Δθ ₁ ) is the difference between the absolute value of the measured value (θ ₁ ) before the start of the analyte contact process and the measured value (θ ₁ ') after the end of the process It is possible to calculate (usually θ ₁ <θ ₁ ′ and Δθ ₁ = θ ₁ ′ −θ ₁ ), and this change amount is used for detection or quantification of the analyte. Such Δθ ₁ reflects the change in dielectric constant accompanying the formation of a thin film by the analyte trapped by the immobilized ligand.

The amount of analyte contained in a sample solution can be quantitatively evaluated by comparing θ ₁ or Δθ ₁ for the sample solution with a reference value obtained using a sample with a known concentration. The relative evaluation can also be made by comparing with θ ₁ or θλ ₁ for other sample solutions.

When θ ₁ does not substantially change, that is, Δθ ₁ is 0 to sufficiently small (within a measurement error range), specific binding between the ligand immobilized on the sensor chip and the analyte in the sample solution occurs. It can be determined that there was no analyte in the dissolved sample solution.

On the other hand, when θ ₁ has changed, that is, when Δθ ₁ is greater than a certain value (measurement error range), an intermolecular interaction has occurred between the ligand immobilized on the sensor chip and the analyte in the sample solution. And it can be determined that the analyte is present in the sample solution. Furthermore, the analyte in the sample solution can be quantified from the degree of change, that is, the magnitude of Δθ ₁ .

<SPFS embodiment>
Similar to the SPR measurement system described above, the SPFS measurement system is mainly provided on the measurement member and the dielectric member side (metal thin film back side) of the measurement member, and the back side of the metal thin film in the measurement region. A light source for irradiating measurement light (incident light) and a light receiver for receiving reflected light of the measurement light, and further provided on the immobilized ligand layer side (metal thin film surface side) of the measurement member for measurement. A photodetector for receiving fluorescence emitted in the region.

In the case of conforming to SPFS, specifically, the analyte can be detected by performing the following steps using the system as described above.
(1) Analyte contact step: a step of bringing a medium containing an analyte into contact with a measurement region where the immobilized ligand layer of the ligand-immobilized sensor chip as described above is formed;
(2) Fluorescence labeling step: a step of bringing a medium containing a labeled ligand into contact with the measurement region that has undergone the above steps;
(3) Signal observation step: a step of irradiating measurement light to the measurement region that has undergone the above steps and observing a signal based on surface plasmon resonance that occurs in the metal thin film.

(Analyte contact process)
Since the analyte contact process in SPFS is the same as the analyte contact process in SPR described above, description thereof is omitted here.

(Fluorescent labeling process)
In the case of SPFS, the medium containing the labeled ligand is brought into contact with the measurement region that has undergone the above-described process, that is, the analyte captured by the ligand-immobilized layer and the labeled ligand are brought into contact with each other, and the immobilized ligand-analyte-labeled ligand A complex is formed.

• Labeled ligand The ligand bound to the fluorophore used for fluorescently labeling the analyte captured on the sensor chip is called “labeled ligand” (“labeled antibody” or “secondary antibody” if the ligand is an antibody). And so on.). The ligand of the labeled ligand (hereinafter sometimes referred to as “second ligand”) and the ligand of the ligand-carrying charged fine particles (hereinafter sometimes referred to as “first ligand”) may be the same or different. Good. However, when the first ligand is a polyclonal antibody, the second ligand may be a monoclonal antibody or a polyclonal antibody, but when the first ligand is a monoclonal antibody, the second ligand is the first ligand It is desirable that the antibody is a monoclonal antibody that recognizes an epitope that is not recognized, or a polyclonal antibody.

  The labeled ligand in SPFS can be prepared in the same manner as a complex (conjugate) of a ligand and a phosphor that is also used in a general immunostaining method. For example, a complex of ligand and avidin (including streptavidin, etc.) and a complex of phosphor and biotin are prepared and reacted to bind the phosphor to the ligand via avidin / biotin. A complex (maximum 4 biotins can bind to 1 avidin) is obtained. In addition to the reaction of biotin and avidin as described above, the primary antibody-secondary antibody reaction mode used in the fluorescent labeling method, carboxyl group and amino group, isothiocyanate and amino group, sulfonyl halide and amino group, Reactions such as iodoacetamide and thiol groups may be used.

-Fluorescent substance "Fluorescent substance" is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or by exciting using the field effect. In the present invention, various known phosphors used in known SPFS or the like or fluorescent labeling methods can be used. Representative phosphors include fluorescent dyes and semiconductor nanoparticles.

  Examples of the fluorescent dye include a fluorescent dye of the fluorescein family (manufactured by Integrated DNA Technologies, such as Fluorescein Isothiocyanate: FITC), a fluorescent dye of the polyhalofluorescein family (manufactured by Applied Biosystems Japan Co., Ltd.), and the hexachlorofluorescein family. Fluorescent dyes (Applied Biosystems Japan Co., Ltd.), Coumarin family fluorescent dyes (Invitrogen Corporation, for example, Aminomethylcoumarin: AMCA), Rhodamine family fluorescent dyes (GE Healthcare Biosciences, For example, Tetramethyl Rhodamine Isothiocyanate: TRITC, Rhodamine Red-X: RRX Texas Red (TR), cyanine family fluorescent dyes (eg, Cy2®, Cy5®, Alexa Fluor® 647), Indian carbocyanine family fluorescent dyes (eg, Cy3®) )), Oxazine family fluorescent dye, thiazine family fluorescent dye, squaraine family fluorescent dye, chelated lanthanide family fluorescent dye, BODIPY (registered trademark) family fluorescent dye (manufactured by Invitrogen Corporation) ), Naphthalenesulfonic acid family fluorescent dye, pyrene family fluorescent dye, triphenylmethane family fluorescent dye, Alexa Fluor (registered trademark) dye series (manufactured by Invitrogen Corporation), and the like. Table 1 shows the absorption wavelength (nm) and emission wavelength (nm) of typical fluorescent dyes included in these families.

In addition to the above organic fluorescent dyes, rare earth complex fluorescent dyes such as Eu and Tb can also be used. In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex-based fluorescent dye is ATBTA-Eu ³⁺ .

  On the other hand, the semiconductor nanoparticles are made of a semiconductor such as Si, Ge, InN, InP, GaAs, AlSe, CdSe, AlAs, GaP, ZnTe, CdTe, InAs, or a raw material for forming them. InP and Si are preferable from the viewpoint of low toxicity, and CdTe and CdSe are preferable from the viewpoint of emission intensity in the visible light range.

Also, semiconductor nanoparticles having a core / shell structure in which the semiconductor nanoparticles are used as a core and a shell is added to the outer layer may be used. As a material for forming the shell, inorganic semiconductors of II-VI group, III-V group, and IV group can be used as in the case of semiconductor nanoparticles having a known core / shell structure. For example, a semiconductor having a larger band gap than each core-forming inorganic material such as Si, Ge, InN, InP, GaAs, AlSe, CdSe, AlAs, GaP, ZnTe, CdTe, InAs, etc. Is preferred. More specifically, for example, ZnS is suitable as the shell when InP, CdTe, and CdSe are used as the core, and SiO ₂ is preferable as the shell when Si is used as the core. It should be noted that the shell may not completely cover the entire surface of the core as long as it does not cause adverse effects even if the core is partially exposed.

  The volume average particle size of the semiconductor nanoparticles not having a core / shell structure (comprising only the core) may be the same as that of the conventional one, but is preferably 1 to 15 nm. The volume average particle diameter of the core / shell structure semiconductor nanoparticle may be the same as that of the conventional one, but is preferably 1 to 20 nm.

  In addition, the particle size of the semiconductor nanoparticles described above and the fluorescence enhancement fine particles (metal colloid particles) and nanoparticle aggregates described below is expressed by a volume average particle size in principle. This volume average particle diameter is measured immediately after the production of each of the above particles (before aggregation) using a particle size measuring apparatus (for example, Malvern Instruments, Zetasizer Nano S) by dynamic light scattering (DLS). The value is obtained by measuring the particle size distribution, and the particle size at the peak (center) position of the particle size distribution is defined as the volume average particle size of the particle.

Such semiconductor nanoparticles can be produced by a known method, and can also be obtained as a product.
(Signal observation process)
In the case of SPFS, the signal based on surface plasmon resonance that occurs in the metal thin film reflects the fluorescence emitted by the phosphor in the complex formed by the fluorescence labeling step. That is, measurement light (excitation light corresponding to a phosphor) is irradiated to the measurement region that has undergone the above steps, and the intensity of fluorescence emitted from the measurement region (assay signal S, unit is au, for example) is obtained as a measurement value. To do. Usually, in order to eliminate the influence of noise and the like, the intensity of the fluorescence (blank signal N, so-called noise) emitted from the measurement region that has not undergone the analyte contact step or the fluorescent labeling step is also obtained as a measurement value. The S / N ratio calculated by the following formula is used for the detection or quantification of the analyte.

S / N ratio = | (assay signal) | / | (blank signal) |
Such an S / N ratio reflects a change in dielectric constant as a thin film is formed by the analyte trapped on the surface of the sensor chip and the labeled ligand bound thereto.

  The amount of analyte contained in a sample solution can be quantitatively evaluated by comparing the S / N ratio for the sample solution with a reference value obtained using a sample with a known concentration, Relative evaluation can also be performed by comparing with the S / N ratio of other sample solutions.

  For example, when the S / N ratio is 1 or close to it (within the measurement error range), specific binding between the ligand immobilized on the sensor chip and the analyte in the sample solution did not occur, that is, the dissolved sample. It can be determined that no analyte was present in the solution.

  On the other hand, when the S / N ratio is larger than a certain value (measurement error range), it is indicated that specific binding between the ligand immobilized on the sensor chip and the analyte in the sample solution has occurred. It can be determined that the analyte is present in the solution. Furthermore, the analyte in the sample solution can be quantified from the size of the S / N ratio.

[Example 1]
A chromium thin film (thickness of 1 to 3 nm) formed by sputtering on a plane of a glass dielectric member, and a gold thin film (thickness of 44 to 52 nm) further formed thereon by sputtering A sensor chip equipped with was prepared.

(Charged layer formation process)
A 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.

For reference, an atomic force microscope (AFM) image of the surface of a cationically modified sensor chip separately prepared by the same method as described above is shown in FIG.
(Immobilized ligand layer formation process)
Immunogold conjugate EM.GMHL 10 (manufactured by BB international) was used as the ligand-supported charged (anion) fine particles. An aqueous solution diluted with 1% by volume was prepared, added to the well having the ionic (cation) -coated sensor chip prepared in the above step as the bottom, shaken for 5 minutes, and then washed. For reference, FIG. 3 shows an atomic force microscope (AFM) image of the surface of a cationically modified sensor chip, to which a ligand-carrying gold fine particle is fixed, prepared separately by the same method as described above.

(Analyte contact process)
IgG was used as an analyte, which was labeled with Alexa Fluor 647 (registered trademark, Invitrogen) to produce a labeled analyte (labeling rate 2.4). A sample solution containing this labeled analyte at a concentration of 10 ng / mL was prepared, and the sensor chip provided in the immobilized ligand layer prepared in the above step was added to the well having the bottom surface and shaken for 5 minutes.

  At the end of each of the charged layer forming step, the immobilized ligand layer forming step, and the analyte contacting step, a sensor chip (measuring member) is attached to the SPFS measuring device and irradiated with measuring light while changing the incident angle. Both the reflection intensity corresponding to the signal in the SPR method and the fluorescence intensity corresponding to the signal in the SPFS method were measured. The results are shown in FIGS. Moreover, the result of having photographed the atomic force microscope (AFM) image after capturing an analyte after completion of the assay is shown in FIG.

[Example 2]
The assay was performed by changing the immobilized ligand layer forming step, the analyte contacting step, and the labeled antibody contacting step of Example 1 as follows.

(Immobilized ligand layer formation process)
“Snowtex XS” (manufactured by Nissan Chemical Industries, Ltd., volume average particle size 5 nm) was used as the immobilized ligand-immobilized SiO ₂ fine particles. 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 the mixture was 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.

  A well member having a circular through hole with a diameter of 5 mm was prepared. This well member was fixed on the surface of the cationically modified sensor chip produced by the above process via matching oil to produce a well-type measurement member.

1 mL of an aqueous solution containing the ligand-carrying charged fine particles (SiO ₂ ) at a concentration of 1% by weight was introduced into the well, shaken for 5 minutes and washed with water to form an immobilized ligand layer.
(Analyte contact process)
A sample containing α-fetoprotein (AFP) (manufactured by Acris Antibodies GmbH) as an analyte in PBS (pH 7.4; manufactured by Nacalai Tesque Co., Ltd.) at a concentration of 1 μg / mL was prepared. This sample is introduced into the closed flow path of the measurement member for 70 minutes at a liquid feed rate of 20 μL / min by the syringe pump (total 1400 μL), and brought into contact with the surface of the sensor chip on which the immobilized ligand layer is formed, The analyte was bound to the immobilized ligand.

(Labeled antibody contact process)
On the other hand, the anti-α fetoprotein mouse monoclonal antibody “6D2” was labeled with Alexa Fluor 647 (registered trademark, Invitrogen) to prepare a labeled analyte (labeling rate 2.5). A sample solution containing this labeled analyte at a concentration of 10 ng / mL was prepared, and the sensor chip provided in the immobilized ligand layer prepared in the above step was added to the well having the bottom surface and shaken for 5 minutes. At the end of each of the charged layer formation step, the immobilized ligand layer formation step, the analyte contact step, and the labeled antibody contact step, a sensor chip (measuring member) is attached to the SPFS measurement device, and measurement is performed while changing the incident angle. Irradiated with light, both the reflection intensity corresponding to the signal in the SPR method and the fluorescence intensity corresponding to the signal in the SPFS method were measured. The results are shown in Table 1.

DESCRIPTION OF SYMBOLS 10 Ligand immobilization sensor chip 100 Sensor base material 102 Dielectric member 104 Metal thin film 120 Charged layer 140 Immobilized ligand layer (ligand carrying charged fine particles)
142 charged fine particle 144 ligand 160 analyte

Claims (9)

A ligand-immobilized localized field optical sensor chip for analyte detection, at least,
A dielectric member;
A metal thin film formed on the dielectric member;
A positively or negatively charged layer formed on the metal thin film;
An immobilized ligand layer composed of a ligand-carrying charged fine particle that is formed on the charged layer and is immobilized by electrostatic interaction and is composed of charged fine particles opposite to the charged layer and a ligand bonded thereto. And a ligand-immobilized localized field photosensor chip.   The sensor chip according to claim 1, wherein the charged layer is a layer made of an ionic polymer.   The sensor chip according to claim 1, wherein the ionic polymer is a polymer having a cationic functional group. The sensor chip according to claim 1, wherein a surface charge density of the charged layer is in a range of 50 to 200 mC / m ² .   The sensor chip according to any one of claims 1 to 4, wherein an arithmetic average roughness of a surface of the charged layer is in a range of 1 to 50 nm.   The sensor chip according to any one of claims 1 to 5, wherein the sensor chip is for SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) or SPR (Surface Plasmon Resonance). The sensor chip described. Wherein the charged particles, SiO _2, TiO _2, is made of an ionic polymer or ligand from fixable metal sensor chip according to any one of claims 1 to 6. A method for producing a ligand-immobilized localized field photosensor chip for analyte detection according to any one of claims 1 to 7,
Charged positively or negatively charged charged layer provided in the local field photosensor chip is contacted with charged fine particles oppositely charged to the charged layer and ligand-carrying charged fine particles composed of ligands bound to the charged fine particles. A production method comprising a step of forming an immobilized ligand layer comprising the ligand-carrying charged fine particles by immobilization by interaction. An analyte detection method using the ligand-immobilized localized field photosensor chip according to any one of claims 1 to 7, comprising at least:
A step of bringing an analyte-containing medium into contact with an immobilized ligand layer in a measurement region of the ligand-immobilized localized field optical sensor chip; and a surface plasmon that occurs in the metal thin film by irradiating the measurement region with the measurement light An analyte detection method comprising a step of observing a signal based on resonance.