JP2013029369A - Ionic functional group-modified local-field optical sensor chip and method for detecting analyte using ligand-carrying charged particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a further highly sensitive detection method of an analyte solving a problem that has been observed in a measuring method using magnetic particles and a magnetic force while effectively using an advantage of combining a homogeneous assay with a heterogeneous sensing (a detecting method of analytes using surface plasmon resonance generated on a metal thin film).SOLUTION: The method for detecting analytes includes the steps for: reacting ligand-carrying charged particles comprising at least charged particles that are charged inversely to a charged layer, and ligand bonded thereto, with analytes in medium, and forming a ligand-carrying charged particle-analyte complex; bringing the medium including the ligand-carrying charged particle-analyte complex into contact with the charged layer in a measuring area of a sensor chip; and applying measuring light to the measuring area and observing a signal based on the surface plasmon resonance generated on the metal thin film.

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.

  In general, such an analyte detection method is a heterogeneous sensing 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. It is. 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.

  INDUSTRIAL APPLICABILITY The present invention is found in a measurement method using magnetic particles and magnetic force while taking advantage of the combination of homogeneous assay and heterogeneous sensing (analyte detection method using surface plasmon resonance occurring in a metal thin film). An object of the present invention is to provide a more sensitive analyte detection method that solves such problems.

  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-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 localized field photosensor chip including at least a dielectric member, a metal thin film formed on the dielectric member, and a positively or negatively charged layer formed on the metal thin film is used. A method for detecting an analyte, at least,
A step of reacting a ligand-carrying charged fine particle composed of charged fine particles opposite to the charged layer and a ligand bonded thereto with an analyte in a medium to form a ligand-carrying charged fine particle-analyte complex. ,
Contacting a medium containing the ligand-carrying charged fine particle-analyte complex with a charged layer in the measurement region of the localized field photosensor chip; and surface plasmon generated in the metal thin film by irradiating the measurement region with measurement light An analyte detection method comprising a step of observing a signal based on resonance.

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

[4] The analyte detection method 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 method for detecting an analyte according to any one of [1] to [4], wherein the arithmetic average roughness of the surface of the charged layer is in the range of 1 to 50 nm.

  [6] The analyte detection method is SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) or SPR (Surface Plasmon Resonance) [1] to [5] ] The method for detecting an analyte 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 fixable metal, [1] - Detection of an analyte according to any one of [6] Method.

  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 analyte detection method according to the present invention, it is possible to detect and quantify the analyte 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 shows the time when the charged layer formation process was completed (“cation” in the graph), the time when the complex contact process was completed (“cation + Ab + Ag” in the graph), and the ligand-carrying charge (anion) used for comparison in Example 1. It is a graph showing the reflection intensity corresponding to the signal in the SPR method, measured at the end point (“cation + Ab” in the graph) of the step of bringing only the fine particles into contact with the charged layer. FIG. 4 shows the time when the charged layer formation process was completed (“cation” in the graph), the time when the composite contact step was completed (“cation + Ab + Ag” in the graph), and the ligand-carrying charge (anion) used for comparison in Example 1. It is a graph showing the fluorescence intensity corresponded to the signal in the PFS method measured at each end point (“cation + Ab” in the graph) of the step of bringing only the fine particles into contact with the charged layer.

  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. In the present invention, 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 used for measurement for detecting 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 substrate”) is generally formed at least on a substrate made of a dielectric member (for example, SiO ₂ ) and on the surface layer side thereof. A thin metal 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 The sensor chip with a charged layer has a surface charge density in order to arrange the ligand-carrying charged fine particles that may form a complex with the analyte at a desired density (preferably close-packed). Is preferably high enough to satisfy at least a certain level. The sensor chip under the conditions in the step of bringing the sensor chip into contact with the charged fine particles, more specifically, under the pH condition of the medium (aqueous solution) containing the ligand-carrying charged fine particles that are complexed with the analyte used in the process. The surface charge density of the (charged layer) can be adjusted, for example, to 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 forming a complex with the analyte 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 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 include SiO ₂ and TiO ₂ , an ionic polymer, and a metal that can fix 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.

  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.

-Analyte detection method-
In the method for detecting an analyte according to the present invention, a sensor chip having a charged layer as described above is used, and a ligand-carrying charged fine particle-analyte complex separately formed is brought into contact therewith.

Such an analyte detection method can be carried out by a procedure according to a conventional analyte detection method except for some steps, and by using a measurement device similar to the conventional one.
-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, SPR, and the like.
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) Complex formation step: Ligand-carrying charged fine particles composed of charged fine particles opposite to those in the charged layer and ligands bound thereto and an analyte are reacted in a medium to form a ligand-carrying charged fine particles-analyte. Forming a light complex;
(2) Complex contact step: a step of bringing an analyte-containing medium into contact with the measurement region where the charged layer is formed of the ionic functional group-modified sensor chip as described above;
(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.

(Composite formation process)
In order to react a ligand-carrying charged fine particle and an analyte in a medium to form a ligand-carrying charged fine particle-analyte complex, generally, a medium containing a ligand-carrying charged fine particle (hereinafter referred to as “ligand-carrying charge”) is used. And a medium containing the analyte (hereinafter referred to as “analyte solution”).

  As these “mediums”, it is preferable to use, for example, water that is widely used in analyte measurement methods, that is, to prepare ligand-carrying charged fine particles 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. It is a medium that can appropriately perform predetermined measurements without interfering with the formation of a complex of ligand-carrying charged microparticles and an analyte and the subsequent specific adsorption between the complex and the charged layer of the sensor chip. If there is a medium other than water, it is also possible to use it.

  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.

In the complex formation step, stirring or the like may be used in combination to promote the formation of the ligand-carrying charged fine particle-analyte complex.
(Composite contact process)
The ligand-carrying charged fine particle-analyte complex (hereinafter sometimes simply referred to as “complex”) is usually prepared by preparing a medium containing the complex (hereinafter also referred to as “sample solution”). The charged layer is brought into contact with 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 trapped by the charged layer and form a thin film.

(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 calculated as the difference between the absolute value of the measured value (θ ₁ ) before the start of the complex contact process and the measured value (θ ₁ ′) after the process ends. It is calculated (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.

In practical measurement according to the present invention, both the ligand-carrying charged fine particle-analyte complex and the unreacted ligand-carrying charged fine particle generated by the complex formation process are usually captured by the charged layer on the surface of the sensor chip. Although a thin film is formed, the amount of change in dielectric constant, that is, the amount of change in measured value (Δθ ₁ ) reflects the mixing ratio thereof.

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.

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, a 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 change in the dielectric constant of the former and the change in the dielectric constant of the latter, the change in the dielectric constant of the entire thin film to be formed, that is, the difference in measured θ ₁ depends on the ratio of the former and the latter. Will occur. 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.

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 .

  In SPR, a predetermined measurement value is obtained continuously before and after the complex contact step as described above, preferably before, during and after the step, and a signal (ie, change in measurement value) is obtained. ) Can be quantified in real time by the ligand-carrying charged fine particle-analyte complex captured on the surface of the sensor chip. 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.

<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) Complex formation step: Ligand-carrying charged fine particles composed of charged fine particles opposite to those in the charged layer and ligands bound thereto and an analyte are reacted in a medium to form a ligand-carrying charged fine particles-analyte. Forming a light complex;
(2) Complex contact step: a step of bringing an analyte-containing medium into contact with the measurement region where the charged layer is formed of the ionic functional group-modified sensor chip as described above;
(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.

Here, in order to measure by SPFS, it is necessary to fluorescently label the analyte by one of the following labeling methods.
In the first labeling method (labeling method A), in the step (1), the analyte is fluorescently labeled, that is, the above-described step is performed using a complex of analyte and phosphor (labeled analyte). (1) is to be performed. Such a labeled analyte can be prepared by a known method, but generally the analyte is reacted with a phosphor in a liquid, so that the analyte can be labeled quickly. preferable. Usually, a labeled analyte is prepared in advance before the step (1).

  The second labeling method (labeling method B) is a step of bringing a medium containing a labeled ligand into contact with the measurement region that has undergone the above step (1) between the above steps (2) and (3) (hereinafter referred to as “fluorescence labeling step”). ").). That is, the ligand-supported charged fine particle-analyte complex is immobilized on the surface of the sensor chip, and then the analyte is fluorescently labeled. Usually, a labeled ligand is prepared in advance before the fluorescent labeling step. The labeled ligand can be prepared by a known method.

-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.
(Composite formation process)
Since the complex formation process in SPFS is the same as the complex formation process in SPR mentioned above, description here is abbreviate | omitted.

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

(Fluorescent labeling process)
When the analyte is fluorescently labeled by the labeling method B, the medium containing the labeled ligand is brought into contact with the measurement region that has undergone the above-described steps, that is, the ligand-carrying charged fine particle-analyte complex captured on the sensor chip surface and The labeled ligand is brought into contact with each other to form a ligand-carrying charged fine particle-analyte-labeled ligand complex.

• 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.

By replacing the ligand with an analyte, a labeled analyte can be produced by the same reaction method.
(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, etc., the intensity of the fluorescence emitted from the measurement region (blank signal N, so-called noise) at a predetermined stage before the signal observation process is also obtained as a measured value and calculated by the following formula: The S / N ratio is used for analyte detection or quantification.

S / N ratio = | (assay signal) | / | (blank signal) |
Such an S / N ratio reflects the number of phosphors contained in the complex formed on the sensor chip surface, that is, the intensity of fluorescence emitted from these phosphors.

  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 around 1 or does not reach a certain value (measurement error range), the complex formed on the surface of the sensor chip does not contain a phosphor for labeling the analyte. That is, it can be determined that the analyte was not present in the dissolved sample solution.

  On the other hand, when the S / N ratio is larger than a certain value (measurement error range), the complex formed on the surface of the sensor chip contains a phosphor for labeling the analyte, and the sample solution contains the analyte. It can be determined that there is a light. 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 the sensor chip using a spin coater (rotation speed: 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.

(Composite formation process)
Immunogold conjugate EM.GMHL 10 (manufactured by BB international) was used as ligand-supported charged (anion) fine particles, and an aqueous solution was prepared by diluting this to 1% by volume.

  On the other hand, IgG was used as an analyte, and this was labeled with Alexa Fluor 647 (registered trademark, Invitrogen) to produce a labeled analyte (labeling rate 2.4), and then this labeled analyte was 5 ng / mL. A sample solution containing a concentration of was prepared.

1 mL of a 1% by volume diluted aqueous solution of the ligand-carrying charged (anion) fine particles and 1 mL of a 5 ng / mL solution of the labeled analyte were mixed and shaken for 10 minutes.
(Composite contact process)
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 in Production Example 1 via matching oil to produce a well-type measurement member.

The reaction solution obtained in the complex formation step was added to a well having a sensor chip including a charged layer prepared in the above step as a bottom, and shaken for 5 minutes.
A sensor chip (measuring member) is mounted on a measuring device for SPFS and irradiated with measuring light while changing the incident angle, and both the reflection intensity corresponding to the signal in the SPR method and the fluorescence intensity corresponding to the signal in the SPFS method are measured. It was measured. For comparison, the measurement was also performed in the case where only ligand-supported charged (anion) fine particles that were not reacted with the analyte and did not form a complex were brought into contact with the charged layer. The results are shown in FIG. 3 and FIG.

  It can be seen from FIG. 3 that the reaction between antiIgG immobilized on gold nanoparticles and an IgG label (analyte) is performed with high efficiency. This indicates that the homogeneous reaction is fast and highly efficient. It can be seen from FIG. 4 that ultrasensitive detection in SPFS can be achieved with the method of the present invention.

[Example 2]
(Ligand-supported charged microparticle production process)
As the anionic polymer fine particles, “Hydran WLS 202” (manufactured by DIC Corporation) was used, and as the ligand, anti-α-fetoprotein [AFP] mouse monoclonal antibody (clone: 1D5; Mikuli Immuno Laboratory Co., Ltd.) was used. . 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.

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

(Composite contact process)
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 in Production Example 1 via matching oil to produce a well-type measurement member.

The reaction solution obtained in the complex formation step was added to a well having a sensor chip including a charged layer prepared in the above step as a bottom, and shaken for 5 minutes.
A sensor chip (measuring member) is mounted on a measuring device for SPFS and irradiated with measuring light while changing the incident angle, and both the reflection intensity corresponding to the signal in the SPR method and the fluorescence intensity corresponding to the signal in the SPFS method are measured. It was measured. The results 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.

DESCRIPTION OF SYMBOLS 100 Sensor base material 102 Dielectric member 104 Metal thin film 120 Charged layer 200 Ligand carrying charged fine particle 202 Charged fine particle 204 Ligand 220 Ligand carrying charged fine particle-analyte complex 222 Analyte

Claims (7)

An analyte using a localized field photosensor chip, comprising at least a dielectric member, a metal thin film formed on the dielectric member, and a positively or negatively charged charged layer formed on the metal thin film A method of detecting, at least,
A step of reacting a ligand-carrying charged fine particle composed of charged fine particles opposite to the charged layer and a ligand bonded thereto with an analyte in a medium to form a ligand-carrying charged fine particle-analyte complex. ,
Contacting a medium containing the ligand-carrying charged fine particle-analyte complex with a charged layer in the measurement region of the localized field photosensor chip; and surface plasmon generated in the metal thin film by irradiating the measurement region with measurement light An analyte detection method comprising a step of observing a signal based on resonance.   The analyte detection method according to claim 1, wherein the charged layer is a layer made of an ionic polymer.   The analyte detection method according to claim 2, 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, the detection method of an analyte according to any one of claims 1 to 3.   The method for detecting an analyte according to any one of claims 1 to 4, wherein the arithmetic average roughness of the surface of the charged layer is in the range of 1 to 50 nm.   The method for detecting an analyte is SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) or SPR (Surface Plasmon Resonance). The method for detecting an analyte according to Item. Wherein the charged particles, SiO _2, TiO _2, is made of an ionic polymer, or the ligand from lockable metal detection method of an analyte according to any one of claims 1 to 6.