JP5541003B2 - Plasmon excitation sensor chip and assay method using the same - Google Patents

Description

  The present invention relates to an assay method for detecting an analyte in a specimen with high sensitivity. More specifically, the present invention relates to an assay method in which an analyte in a specimen immobilized by contacting the specimen with a substrate is detected with high sensitivity using chemiluminescence measurement utilizing plasmon excitation.

  With surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), irradiation is performed by generating a dense wave (surface plasmon) on the surface of the metal thin film under the condition that the irradiated laser light is attenuated by total reflection (ATR) on the gold thin film surface. The amount of photons that the laser beam has is increased to several tens to several hundreds times (electric field enhancement effect of surface plasmons), and by this, the fluorescent dye in the vicinity of the metal thin film is efficiently excited. It is a method that can detect an analyte. For this reason, in order to increase the sensitivity of bioassays, various researches and developments of sensors and assay methods based on SPFS have been conducted in recent years.

  On the other hand, a chemiluminescence assay using singlet oxygen is known as a measurement method capable of performing measurement in real time during the binding reaction between an analyte and a fluorescent dye. A representative example is the LOCI method (Luminescent Oxygen Channeling Immunoassay) developed by Ullman et al. Of Dade Behring (currently Siemens Healthcare). This LOCI method is a method for detecting the presence of an analyte in a specimen based on the presence or absence of chemiluminescence using particles having a photosensitizer (Sensi beads) and particles having a chemiluminescent compound (Chem beads). (Patent Document 1, Non-Patent Document 1). In this LOCI method, sensi beads and chemi beads are bound to each other by an analyte in a specimen, and photosensitizers on the sensi beads are excited by irradiation of incident light to generate singlet oxygen. It is based on the reaction with the chemiluminescent compound above to form a chemiluminescent active molecular species that fluoresces when it further transitions to a more stable ground state. This LOCI method has already been implemented as a practical system by PerkinElmer, and Sensi beads and chemi beads are also commercially available.

  In addition, the assay system based on this LOCI method is configured to detect a signal due to chemiluminescence in a region shorter than the excitation wavelength of the photosensitizer, for example, 520 to 620 nm by irradiation with excitation light of 680 nm. A configuration for detecting chemiluminescence is employed. Accordingly, the detection light is not limited to the wavelength in the red to near-infrared region, and the wavelength in the green region can be adopted. Therefore, there is an advantage that a wide variety of light detection systems such as a CCD can be selected.

  In addition, due to the molecular lifetime of singlet oxygen, chemiluminescence assays using singlet oxygen such as the LOCI method measure chemiluminescence in real time during the binding reaction between the analyte and the particles containing the chemiluminescent compound. It is possible to do. In the case of the LOCI method, it is said that light emission occurs only when the sensi beads and the chemi beads are close to each other (less than 200 nm).

  Further, as a measuring apparatus to which the LOCI method can be applied, a mixing apparatus for assay reaction in which many closed cuvettes are arranged, and a measuring apparatus having a light receiving system using a light source and a photomultiplier tube using an optical fiber are disclosed in Patent Document 2. It is disclosed in FIG.

  However, all of the known chemiluminescence assay methods using singlet oxygen such as the above-mentioned LOCI method are used in homogeneous systems in solution or suspension. There is no known application or combination with signal enhancement by plasmon excitation.

Japanese Patent No. 3892912 Japanese National Patent Publication No. 9-510656

Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994 Biochemistry

  In the assay method by SPFS, the electric field enhancement effect generated in the metal thin film extends to the range of about 200 nm from the surface of the metal thin film. Therefore, not only the fluorescent dye immobilized on the surface of the metal thin film through the analyte, A fluorescent dye that is not fixed to the substrate is also excited to generate fluorescence. For this reason, after the completion of the binding reaction between the analyte immobilized on the metal thin film surface and the fluorescent dye, a step of washing and removing the unreacted fluorescent dye not immobilized on the metal thin film surface is required prior to the fluorescence measurement. ing. For this reason, the assay method using SPFS has a drawback that it is difficult to perform measurement in real time during the binding reaction between the analyte and the fluorescent dye. In addition, since the washing is performed after the binding reaction between the analyte and the fluorescent dye, the detection fluorescent signal is reduced due to a decrease in the amount of the fluorescent dye fixed on the surface of the metal thin film, and the detection fluorescent signal is not uniform. There is. The fact that only the fluorescence signal at the time after washing can be detected also affects the reliability of the detection result. Furthermore, these problems also mean that the measurement takes a certain amount of time. Also, from the viewpoint of detection wavelength, it is necessary to set the detection wavelength in the near-infrared region because the SPFS assay method often uses gold having absorption in the visible wavelength region for the metal thin film. In many cases, there are restrictions on the types of detection devices that can be used as the chemiluminescence detector.

  For this reason, it is possible to perform measurement in real time during the binding reaction between the analyte and the fluorescent dye while obtaining the benefit of high-sensitivity measurement due to the electric field enhancement effect of surface plasmons, and a highly reliable assay method Is desired.

  In addition, the known chemiluminescence assay methods using singlet oxygen such as the LOCI method have a drawback that it is difficult to obtain sufficient detection sensitivity when used as an assay method because of low luminescence intensity. is there. When measuring in a homogeneous system as in the LOCI method, only chemiluminescence from the surface of the chemibead facing the chemiluminescence detector is the target of measurement, and the detection efficiency of the generated chemiluminescence is constant. There are limits. In addition, in detecting chemiluminescence, it is necessary to remove noise caused by incident light from the light source, but in many cases, it is difficult to completely remove noise caused by incident light from the light source using a filter or the like. is there. Therefore, it is necessary to detect chemiluminescence after completing irradiation of incident light using time-resolved measurement, as in the system put into practical use by PerkinElmer, which not only complicates the measurement procedure but also detects it. There is also a problem that is disadvantageous in terms of sensitivity. In addition, in the LOCI method, not only the above-mentioned chemibeads but also the above-mentioned sensibeads are particles made of resin or the like, so the chembeads may adsorb nonspecifically with the sensibeads, and there is a certain limit to the accuracy of the detection signal is there.

  In order to overcome such problems, the present invention combines a chemiluminescence assay method using singlet oxygen with an assay method using surface plasmon excitation, while having high sensitivity and accuracy. An object of the present invention is to provide an assay method that enables real-time measurement and detection with green light.

  As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention have adopted an electric field enhancement effect by surface plasmon excitation by incorporating a measurement principle based on chemiluminescence such as LOCI method in an assay method utilizing surface plasmon excitation. It has been found that there is also a benefit that real-time measurement based on the principle of chemiluminescence using singlet oxygen is possible, and the present invention has been completed.

That is, the plasmon excitation sensor chip of the present invention is
A transparent dielectric substrate;
A metal thin film;
A layer containing a photosensitizer (photosensitizing layer) in this order ,
The photosensitizing layer comprises a singlet oxygen generator; and
A layer made of a dielectric is provided between the metal thin film and the photosensitizing layer .

In one preferred embodiment of the plasmon excitation sensor chip of the present invention, the photosensitizing layer further includes a dielectric.

The plasmon excitation sensor of the present invention is characterized in that a ligand is bound to the surface of the plasmon excitation sensor chip on which the photosensitizing layer is present, and typically the ligand is an antibody. .

In addition, the assay method of the present invention comprises:
Step (a): A step of bringing a specimen into contact with the plasmon excitation sensor to obtain a specimen-fixed plasmon excitation sensor;
Step (b): A chemiluminescent molecule-immobilized plasmon excitation sensor is obtained by bringing a chemiluminescent molecule-modified ligand complex comprising a second ligand and a chemiluminescent molecule into contact with the analyte-immobilized plasmon excitation sensor and reacting them. Process;
Step (c): Excitation of the chemiluminescence excited by irradiating the chemiluminescent molecule-fixed plasmon excitation sensor with a laser beam from a surface of the transparent dielectric substrate opposite to the metal thin film via a prism. Measuring the amount of fluorescence emitted from the molecule; and
Step (d): The method includes a step of calculating the amount of the analyte contained in the sample from the measurement result obtained in the step (c).

In the assay method of the present invention, the chemiluminescent molecule is usually a molecule that emits chemiluminescence in the presence of singlet oxygen.
In a desirable embodiment of the assay method of the present invention, the chemiluminescent molecule-modified ligand complex is a microparticle modified with a chemiluminescent molecule and a second ligand.

In the assay method of the present invention, the specimen is typically at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva.
The present invention also provides:
Plasmon excitation sensor chip;
A lysate or diluent for lysing or diluting the specimen;
A solution used in the reaction between the plasmon excitation sensor chip and the specimen; and
Also provided is a kit comprising a cleaning reagent.

  The present invention can increase the singlet oxygen generation efficiency by combining surface plasmon excitation and chemiluminescence, and can use a measurement system similar to the measurement system normally used in SPFS. Assay methods can be provided. Also, real-time measurement and detection in the green region, which were difficult with SPFS, are possible. Furthermore, it is not necessary to apply complex surface modifications such as avidin and biotin on the surface of the carrier where the photosensitizer is present, and nonspecific adsorption of chemiluminescent molecules to the photosensitizer can be suppressed. It is possible to provide a more accurate assay method that is easier to operate than the method.

  In addition, since the chemiluminescence can be measured while flowing the liquid through the flow path, the oxygen contained in the liquid can be sufficiently brought into contact with the photosensitizing layer formed on the sensor. Thus, singlet oxygen generated more efficiently can be sufficiently brought into contact with the chemiluminescent molecules immobilized on the sensor, so that detection with higher sensitivity can be performed.

It is a schematic diagram showing a plasmon excitation sensor and assay method according to the present invention.

Hereinafter, the present invention will be specifically described with reference to FIG.
[Plasmon excitation sensor]
The plasmon excitation sensor chip 11 according to the present invention includes a transparent dielectric substrate 111, a metal thin film 112, and a layer containing a photosensitizer (hereinafter referred to as “photosensitized layer”) 113 in this order. And

<Transparent dielectric substrate>
In the present invention, a transparent dielectric substrate 111 is used as a dielectric substrate that supports the structure of the plasmon excitation sensor chip 11. In the present invention, the transparent dielectric substrate 111 is used as the dielectric substrate because light irradiation to the metal thin film 112 described later is performed through the dielectric substrate.

  As will be described later, since the metal thin film 112 is formed on the surface of the transparent dielectric substrate 111, the transparent dielectric substrate 111 used in the present invention has at least a metal thin film forming plane. An example of such a transparent dielectric substrate 111 is a transparent flat substrate. The transparent dielectric substrate 111 used in the present invention may further include other components such as a prism portion and a sample holding portion in addition to the metal thin film forming plane. For example, an integrated structure block including a metal thin film forming plane, a prism portion, and a sample holding portion may be used.

The material for the transparent dielectric substrate 111 used in the present invention is not particularly limited as long as the object of the present invention is achieved. For example, the transparent dielectric substrate 111 may be made of glass, or may be made of an optical resin such as polycarbonate (PC), polymethyl methacrylate (PMMA), or cycloolefin polymer (COP).
The transparent dielectric substrate 111 has a refractive index at the d-line (588 nm) [n _d] is preferably 1.40 to 2.20.

(1) Transparent flat substrate The transparent flat substrate used as the transparent dielectric substrate 111 in the present invention is usually made of glass or an optical resin such as polycarbonate (PC), polymethyl methacrylate (PMMA), or cycloolefin polymer (COP). It is a transparent flat substrate made of steel.

When using a glass flat transparent substrate as a transparent dielectric substrate 111, commercially, SCHOTT AG Corp. BK7 (refractive index [n _d] 1.52) and LaSFN9 (refractive index [n _d] 1.85) , Ltd. Sumita Optical Glass manufactured K-PSFn3 (refractive index [n _d] 1.84), K-LaSFn17 (refractive index [n _d] 1.88) and K-LaSFn22 (refractive index [n _d] 1 .90) and S-LAL10 (refractive index [n _d ] 1.72) manufactured by OHARA INC. Are preferable from the viewpoint of optical properties and detergency.

  With respect to the transparent flat substrate used as the prism portion transparent dielectric substrate 111, the size (vertical × horizontal) is not particularly limited as long as the thickness is preferably 0.01 to 10 mm, more preferably 0.5 to 5 mm.

The transparent flat substrate used as the transparent dielectric substrate 111 is preferably cleaned with acid and / or plasma before the metal thin film 112 is formed on the surface.
As the cleaning treatment with an acid, it is preferable to immerse in 0.001 to 1N hydrochloric acid for 1 to 3 hours.
Examples of the plasma cleaning treatment include a method of immersing in a plasma dry cleaner (PDC200 manufactured by Yamato Scientific Co., Ltd.) for 0.1 to 30 minutes.

(2) Integrated Structure Block The integrated structure block used as the transparent dielectric substrate 111 in the present invention includes a metal thin film forming plane, a prism portion, and a sample holding portion. The integrated structure block has a structure in which the metal thin film forming plane and the prism portion are integrated, and the metal thin film forming plane is located on the light reflecting surface of the prism portion. Further, the integrated structure block has a side surface structure continuously formed so as to surround the metal thin film forming plane, and a recess formed by the side surface structure and the metal thin film forming plane is a sample holding portion. Is configured. That is, the metal thin film forming plane constitutes the bottom surface of the sample holder, and the side structure constitutes the side surface of the sample holder. Advantages of using an integrated structure block as the transparent dielectric substrate 111 in the present invention are that the plasmon excitation sensor chip can be easily replaced, and therefore, it is possible to increase the throughput of analyte detection, and a metal thin film The structure in which the forming plane and the prism portion are integrated is that the reflection of incident light at the interface between the metal thin film forming plane and the prism portion can be suppressed.

  This integrated structure block is usually a block made of glass or resin. In particular, optical resin blocks such as polycarbonate (PC), polymethylmethacrylate (PMMA), and cycloolefin polymer (COP) have an integrated structure for reasons such as price, moldability, and low optical properties due to molding. It is suitably used for body block applications. The integrated structure block used as the transparent dielectric substrate 111 can be formed, for example, by injection molding a raw material resin, or ZEONEX (registered trademark) 330R (refractive index 1.50) manufactured by Nippon Zeon Co., Ltd. Commercial products such as these can also be used. In the integrated structure block used in the present invention, the size is not particularly limited because the metal thin film forming plane and the prism portion are integrated.

  Even when an integrated structure block is used as the transparent dielectric substrate 111, it is preferable to clean the surface with acid and / or plasma before forming the metal thin film 112 on the surface. The conditions for the acid cleaning process and the plasma cleaning process are the same as in the case where a transparent flat substrate is used as the transparent dielectric substrate 111.

《Metal thin film》
In the plasmon excitation sensor chip 11 according to the present invention, a metal thin film 112 is formed on one surface of a transparent dielectric substrate 111. The metal thin film 112 has a role of generating surface plasmon excitation by irradiation light from a light source. In the present invention, the surface plasmon excitation generated in the metal thin film 112 excites a photosensitizer contained in the photosensitizing layer 113 described later to generate an active molecule, and this active molecule and the chemiluminescent molecule 22 described later Chemiluminescence is generated through this reaction.

  The metal thin film 112 formed on one surface of the transparent dielectric substrate 111 is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and has chemical stability. From the viewpoint of optical properties, it is more preferable to be made of gold. About these metals, the form of the alloy may be sufficient and what laminated | stacked the some metal may be sufficient. Such a metal species is preferable because it is stable against oxidation and is highly excited by surface plasmons.

  When a glass flat substrate is used as the transparent dielectric substrate 111, it is preferable to form a thin film of chromium, nickel chromium alloy or titanium in advance in order to bond the glass and the metal thin film 112 more firmly.

  Examples of the method for forming the metal thin film 112 on the transparent dielectric substrate 111 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 the chromium thin film and / or the metal thin film 112 by sputtering or vapor deposition.

  The thickness of the metal thin film 112 is preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 to 500 nm, and alloys thereof: 5 to 500 nm. The thickness of the chromium thin film is preferably 1 to 20 nm.

  From the viewpoint of the electric field enhancing 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, and chromium The thickness of the thin film is more preferably 1 to 3 nm.

  It is preferable that the thickness of the metal thin film 112 is within the above range because surface plasmons are likely to be generated. Moreover, as long as the metal thin film 112 has such a thickness, the size (length × width) is not particularly limited.

《Photosensitizing layer》
In the plasmon excitation sensor chip 11 according to the present invention, a photosensitizing layer 113 is formed on the surface of the metal thin film 112. This photosensitizing layer 113 is a layer containing a photosensitizer, and receives active surface molecules 41 that can excite chemiluminescent molecules 22 to be described later by receiving surface plasmons generated on the metal thin film 112 by light irradiated from a light source. Has the role of generating

  The active molecule 41 generated by the photosensitizer contained in the photosensitizing layer 113 is a molecule that generates chemiluminescence by binding to the chemiluminescent molecule 22 and generating a chemiluminescent molecular species. However, in the present invention, the active molecule 41 generated by the photosensitizer excites the chemiluminescent molecule 22 immobilized on the plasmon excitation sensor chip 11, but modifies the chemiluminescent molecule that exists in the solution in a free state. The chemiluminescent molecule 22 contained in the ligand complex 20 is required to be an active molecule that is not excited, and examples of such an active molecule include a metastable molecule having a finite molecular lifetime such as singlet oxygen. . From this, a singlet oxygen generator is mentioned as a typical example of the photosensitizer used in the present invention. As singlet oxygen generators, phthalocyanines, porphyrins, methylene blue (red functional ones), rose bengal (green functional ones), endoperoxide, fullerene C60, rubrene (single oxygen generators in the presence of yellow to ultraviolet light) Among them, dyes such as phthalocyanine, methylene blue, and rose bengal are preferably used in the present invention. The triplet state of these dye molecules has an excitation energy approximately equal to the energy difference between singlet oxygen and triplet oxygen. Therefore, when these dyes are photoexcited by light irradiation, transferred to the triplet state by intersystem crossing, and the triplet state dye molecule collides with triplet oxygen, electrons and energy are generated between the dye and oxygen. Exchange occurs, and at the same time the dye returns to the ground state, triplet oxygen transitions to singlet oxygen. In this way, singlet oxygen is generated by light irradiation of these dyes.

  The plasmon excitation sensor chip 11 according to the present invention is used for an assay method in a system using a measurement system for SPFS. Here, since gold is frequently used as the metal thin film 112, a dye that generates singlet oxygen by plasmon excitation by 650-680 nm incident light is preferably used in order to influence the light absorption by gold. From this point, phthalocyanine is particularly preferably used.

The thickness of the photosensitizing layer 113 in the present invention is usually 5 nm or more and 50 nm or less from the viewpoint of an enhanced electric field and metal quenching.
As a method for forming the photosensitizing layer 113, a conventionally known method such as vapor deposition or application of a photosensitizer or an appropriate composition containing the photosensitizer on the metal thin film 112 may be used.

<Dielectric layer>
The plasmon excitation sensor chip 11 according to the present invention provides a foundation for fixing a ligand to be described later on the plasmon excitation sensor chip 11 and also provides a dielectric to provide smoothness and durability to the surface of the plasmon excitation sensor chip 11. It is preferable to have a layer (hereinafter referred to as a “dielectric layer”).

  In one embodiment, the dielectric layer is present as a photosensitizing layer 113 containing a dielectric (hereinafter “dielectric-containing photosensitizing layer”). In this case, in the plasmon excitation sensor chip 11, the photosensitizing layer 113 further includes a dielectric.

  In another embodiment, the dielectric layer may be present as a layer different from the photosensitizing layer 113. However, the active molecule 41 such as singlet oxygen is usually activated by a photosensitizer contained in the photosensitizing layer 113, for example, a precursor component contained in the solution, for example, dissolved oxygen present as triplet oxygen. Therefore, the photosensitizing layer 113 needs to be present at a position where it can come into contact with the solution. Therefore, when the dielectric layer is present as a layer different from the photosensitizing layer 113, the dielectric layer is usually under the photosensitizing layer 113 so as not to impair the function of the photosensitizing layer 113. That is, it exists between the metal thin film 112 and the photosensitizing layer 113 (such a dielectric layer is referred to as a “photosensitive layer-underlying dielectric layer”). The dielectric layer under the photosensitizing layer may be formed in combination with the photosensitizing layer 113 that does not contain a dielectric, or the photosensitizing layer 113 that contains a dielectric (that is, the dielectric-containing photosensitizing layer described above). It may be formed in combination with a photosensitive layer. When the dielectric layer containing the photosensitizing layer is provided, the thickness is usually 5 nm to 1000 nm, although the thickness varies depending on the thickness of the photosensitizing layer and the type of dielectric used.

  Further, in an aspect in which the dielectric-containing photosensitizing layer and the dielectric-containing photosensitizing layer are combined, the dielectric-containing layer has a three-dimensional network skeleton structure and a three-dimensional network pore structure. It may be a photosensitizing layer 113 having a form in which a photosensitizer is bonded to a porous dielectric layer (hereinafter referred to as “porous photosensitizing dielectric layer”). In such a porous photosensitized dielectric layer, the photosensitizer is present in layers, for example, on the surface of the porous dielectric layer, inside the pore structure of the porous dielectric layer, or both. Yes. In an embodiment in which the dielectric layer is such a porous photosensitized dielectric layer, the dielectric layer can fix a large number of ligands 12 to be described later, which is advantageous in performing high sensitivity measurement. . In addition, since the dielectric layer has a flow path-like structure inside, the dissolved oxygen in the solution supplied through the flow path is not only the photosensitizer present on the surface of the layer, but also this flow-path-like structure. In addition, there is also an advantage that the photosensitizer contained in the layer can be efficiently contacted through the active layer 41, whereby the active molecules 41 can be generated more efficiently. The thickness of such a porous photosensitized dielectric layer may vary from 400 nm to 2 μm, more preferably from 400 nm to 1 μm, although it varies depending on the type of dielectric used.

  Examples of the dielectric constituting such a dielectric layer include inorganic dielectrics such as titania and silica, and organic dielectrics such as organic polymers. When the plasmon excitation sensor chip 11 is used as the plasmon excitation sensor 20, an inorganic dielectric, particularly titania, silica or the like is preferably used from the viewpoint that there is little noise in the measurement signal. In the case where the photosensitizing layer 113 is the porous photosensitizing dielectric layer, it may be an inorganic dielectric having a porous structure, a fine particle inorganic dielectric, a binder material made of a hydrophilic polymer, and the like. The porous structure which consists of may be sufficient.

  This dielectric layer can also be formed in the form of a dielectric-containing photosensitizing layer by depositing or coating an appropriate composition containing a photosensitizer and a dielectric on the surface of the metal thin film 112. Alternatively, before the photosensitizing layer 113 is formed, a dielectric material may be applied to the surface of the metal thin film 112 to form a dielectric layer under the photosensitizing layer. In addition, a basic porous dielectric formed by forming an inorganic dielectric having a porous structure on the surface of the metal thin film 112 or a mixed liquid of a fine particle inorganic dielectric and a binder substance is applied to the surface of the metal thin film 112. A porous photosensitized dielectric layer may be formed by bringing a solution containing a photosensitizer into contact with the obtained basic porous dielectric. In any case, a functional group such as an amino group, a thiol group, or a carboxyl group is present on the surface of the dielectric layer as a ligand fixing binding group for fixing the ligand 12 described later to the plasmon excitation sensor chip 11. You may do it.

<< Method of manufacturing plasmon excitation sensor chip >>
In the plasmon excitation sensor chip 11 according to the present invention, a metal thin film 112 is formed on the surface of a transparent dielectric substrate 111, and then a photosensitizer or a suitable composition containing a photosensitizer is formed on the surface of the metal thin film 112. It can be obtained by forming the photosensitizing layer 113 by vapor deposition or coating. Here, by using a mixture of a photosensitizer and a dielectric as a composition containing a photosensitizer, a plasmon having a photosensitizing layer 113 containing a dielectric (that is, a dielectric-containing photosensitizing layer). The excitation sensor chip 11 can be obtained. Alternatively, the plasmon excitation sensor chip 11 having the dielectric layer under the photosensitizing layer can be obtained by applying a dielectric to the surface of the metal thin film 112 before forming the photosensitizing layer 113. .

[Plasmon excitation sensor]
The plasmon excitation sensor 10 according to the present invention is characterized in that a ligand is bonded to the surface of the plasmon excitation sensor chip 11 on which the photosensitizing layer 113 is present.

<Ligand>
In the plasmon excitation sensor 10 according to the present invention, the ligand 12 is bonded to the surface of the plasmon excitation sensor chip 11 described above on which the photosensitizing layer 113 is formed. The ligand 12 is used for the purpose of fixing the analyte 31 in the specimen to the plasmon excitation sensor 10.

  In the present invention, “ligand” refers to a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to an analyte contained in a specimen. Examples of such “molecule” or “molecular fragment” include nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples include, but are not limited to, molecules and complexes.

  Examples of the “protein” include antibodies and the like, specifically, anti-α-fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory), anti-carcinoembryonic antigen (CEA) ) Monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody and the like.

In the present invention, the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments.
When an antibody is used as the ligand 12 in the present invention, the ligand may also be used in step (b) in the assay method described below for the purpose of binding the chemiluminescent molecule 22 to the analyte 31. Therefore, in the present invention, a ligand that is immobilized in advance on the plasmon excitation sensor 10 may be referred to as a “first ligand”, and a ligand that is used in step (b) described later may be referred to as a “second ligand”.

《Ligand fixation mode》
In the plasmon excitation sensor 10 of the present invention, the mode of binding of the ligand 12 to the plasmon excitation sensor chip 11 is not particularly limited.

  As a typical binding mode, there is a mode in which the ligand 12 itself is bound to the surface of the photosensitizing layer 113 or the dielectric layer. For example, when the photosensitizing layer 113 or the dielectric layer already has the ligand-fixing binding group, the ligand 12 is directly bonded to the surface of the photosensitizing layer 113 and the dielectric layer by a conventional method. Can be made. On the other hand, when the dielectric layer does not have the ligand-fixing binding group, such as when the dielectric layer is formed only from the inorganic dielectric, an appropriate ligand-fixing binding group is used. The ligand-fixing binding group is introduced into the dielectric layer by allowing the silane coupling agent to act, and then the ligand 12 is attached to the surface of the dielectric layer through the ligand-fixing binding group by a conventional method. It may be fixed to. The ligand-fixing binding group introduced here is the same as the ligand-fixing binding group described above in the section of the dielectric layer. However, when a silane coupling agent having a ligand-fixing binding group is allowed to act, it is preferable to appropriately adjust the reaction position and concentration of the silane coupling agent as necessary.

[Assay Method]
The assay method according to the present invention comprises:
Step (a): A step of bringing a specimen into contact with the plasmon excitation sensor 10 to obtain a specimen-fixed plasmon excitation sensor;
Step (b): The analyte-immobilized plasmon excitation sensor is brought into contact with a chemiluminescent molecule-modified ligand complex 20 composed of the second ligand 21 and the chemiluminescent molecule 22, and these are reacted to excite the chemiluminescent molecule-immobilized plasmon. Obtaining a sensor;
Step (c): Excited chemiluminescent molecules by irradiating the chemiluminescent molecule-fixed plasmon excitation sensor with laser light from the surface of the transparent dielectric substrate 111 opposite to the metal thin film 112 via a prism. Measuring the amount of fluorescence emitted from 22; and
Step (d): The method includes a step of calculating the amount of the analyte 31 contained in the specimen from the measurement result obtained in the step (c).

<Process (a)>
In the assay method of the present invention, step (a) is a step of obtaining a specimen-fixed plasmon excitation sensor by bringing the specimen into contact with the plasmon excitation sensor 10 described above.

  When the analyte 31 to be measured by the assay method of the present invention is contained in the specimen, the analyte 31 is fixed to the plasmon excitation sensor 10 via the ligand 12 as shown in FIG. In the present invention, the plasmon excitation sensor 10 to which the analyte 31 in the sample is thus bonded is referred to as “sample fixed plasmon excitation sensor”.

Specimen In the present invention, “specimen” refers to various samples to be measured by the assay method of the present invention.

Examples of the “specimen” include blood (serum / plasma), urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc., and appropriately diluted in a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred. These may be used alone or in combination of two or more.
When the analyte to be detected in the detection method of the present invention is a nucleic acid or the like, a sample into which chemiluminescent molecules 22 described later are introduced in advance may be used as the “sample”.

"Analyte" in analyte present invention are specifically recognized the first ligand 12 immobilized on the surface of the photosensitizer layer 113 or含誘collector layer (or recognized) molecules capable of binding Or it means a molecular fragment. Examples of such “molecule” or “molecular fragment” include, for example, nucleic acid (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), which may be single-stranded or double-stranded, or the like, or Nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof In particular, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, or the like, and is not particularly limited.

Contact In the present invention, “contact” means that a surface on which a ligand 12 or the like of a plasmon excitation sensor is immobilized is immersed in a liquid supply, and an object contained in the liquid supply is excited by the plasmon excitation. Contacting a sensor. By this “contact”, an analyte-ligand complex in which the ligand 12 and the analyte 31 are combined is formed. In the step (a), the “contact” between the specimen and the plasmon excitation sensor means that the specimen is included in the liquid feed circulating in the flow path, and only the one surface on which the ligand of the plasmon excitation sensor is immobilized is the liquid feed. A mode in which the plasmon excitation sensor and the specimen are brought into contact with each other in a state of being immersed therein is preferable.

  The “channel” is a rectangular parallelepiped or a tube that can efficiently deliver a small amount of a chemical solution, and can change or circulate the solution feeding speed in order to promote the reaction. . Here, as the shape of this flow path, the vicinity of the location where the plasmon excitation sensor is installed preferably has a rectangular parallelepiped structure, and the vicinity of the location where the drug solution is delivered preferably has a tubular shape.

  The material is a homopolymer or copolymer containing methyl methacrylate, styrene or the like as a raw material in the plasmon excitation sensor part; a light-transmitting material such as polyolefin such as polyethylene, and a silicone rubber, Teflon ( (Registered trademark), polymers such as polyethylene and polypropylene are used.

  However, for the plasmon excitation sensor unit, the flow channel structure is not necessarily made of a light-transmitting material unless the flow channel is kept in a fixed shape at the time of fluorescence measurement and the detection of fluorescence generated by plasmon excitation is not hindered. There is no need to consist only of. That is, of the flow path of the plasmon excitation sensor part, a part necessary for transmitting the fluorescence generated by plasmon excitation and guiding it to the detection part, specifically, a part including a transparent window necessary for collecting the fluorescence. However, other parts may be partially or entirely made of a chemically stable material other than the light transmissive material. When the flow path of the plasmon excitation sensor unit has a rectangular parallelepiped structure, when the surface of the plasmon excitation sensor 20 where the photosensitizing layer 113 exists on the surface is the bottom surface, for example, the ceiling surface at a position facing the bottom surface is The light transmitting material may be used, and the side surface may be formed of a chemically stable material other than the light transmitting material.

  Here, the other part, for example, the side surface does not necessarily need to be a rigid body as long as a certain shape is maintained at the time of fluorescence measurement, and may have an appropriate elasticity to ensure a sealing property. For example, regarding the flow path of the plasmon excitation sensor unit, the ceiling surface may be made of polymethyl methacrylate (PMMA) and the side surface may be made of silicone rubber.

  In the plasmon excitation sensor unit, from the viewpoint of increasing the contact efficiency with the specimen and shortening the diffusion distance, it is preferable that the cross section of the channel of the plasmon excitation sensor unit is independently about 100 nm to 1 mm in length and width.

  In the flow path, the position of the liquid feeding inlet for introducing the liquid feeding from the drug delivery section to the plasmon excitation sensor section and the position of the liquid feeding outlet for discharging the liquid feeding from the plasmon excitation sensor section are both obstructing the fluorescence measurement. Unless it becomes, it will not specifically limit. For example, when the flow path of the plasmon excitation sensor unit has a rectangular parallelepiped structure, it is convenient in creating the flow path to provide both the liquid feeding inlet and the liquid feeding outlet on the ceiling surface. Either one or both of the liquid discharge ports may be provided on the side surface.

The method for fixing the plasmon excitation sensor to the flow path is not particularly limited as long as the flow path is kept in a fixed shape and the fluorescence measurement is not hindered.
As an example of such a fixing method, in a small lot (laboratory level), first, a silicone rubber sheet having a certain thickness on the surface on which the photosensitizing layer 113 of the plasmon excitation sensor is formed. Alternatively, a side structure of the flow path is formed by placing an O-ring, and then a light-transmitting top plate (for example, a PMMA substrate) provided with a liquid feeding inlet and a liquid feeding outlet is disposed thereon. A method of forming the ceiling surface of the flow path by the above, then crimping them and fixing them with an appropriate fastener, etc. At this time, if a silicone rubber sheet having an appropriate thickness and having a hole having an arbitrary shape and size is used as a material constituting the side surface structure, the inner periphery of the hole has a plasmon. Since it becomes the side structure of the flow path of an excitation sensor part, since the flow path which has a required shape and a size can be formed easily, it is preferable. For example, first, a perforated polydimethylsiloxane (PDMS) sheet having a flow path height of 0.5 mm is formed on the surface of the plasmon excitation sensor on which the photosensitizing layer 113 is formed. Next, a PMMA substrate provided with a liquid feeding inlet and a liquid feeding outlet in advance is placed on the polydimethylsiloxane (PDMS) sheet, and then placed. The PMMA substrate, the polydimethylsiloxane (PDMS) sheet, and the plasmon excitation sensor are pressure-bonded and fixed with a fastener such as a screw. In addition, when a silicone rubber sheet or O-ring and a light-transmitting top plate are pressure-bonded and fixed to the plasmon excitation sensor, an appropriate spacer made of a material such as silicone rubber or stainless steel is attached as necessary. You may use together.

  Also, in large lots (factory level) manufactured industrially, as a method of fixing the plasmon excitation sensor to the flow path, a gold substrate is directly formed on a plastic integrally molded product, or a separately manufactured gold substrate is fixed. Then, after the dielectric layer, the SAM layer, and the ligand are immobilized on the gold surface, it can be manufactured by covering with a plastic integrally molded product corresponding to the top plate of the flow path. If necessary, the prism can be integrated into the flow path.

  When an integrated structure block is used as the transparent dielectric substrate 111, since the side structure is already formed as the sample holding portion, a liquid supply inlet and a liquid discharge outlet are provided on the integrated structure block. Placing the light-transmitting top plate (for example, PMMA substrate) to form the ceiling surface of the flow path, and then crimping them and fixing them with appropriate fasteners to plasmon excitation The sensor can be fixed.

  The “liquid feeding” is preferably the same as the solvent or buffer in which the specimen is diluted, and examples thereof include phosphate buffered saline (PBS) and Tris buffered saline (TBS), but are not particularly limited. It is not something.

  The temperature and time for circulating the liquid supply vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. × 1 to 60 minutes, preferably 37 ° C. × 5 to 15 minutes.

The initial concentration of the analyte contained in the sample being sent may be 100 μg / mL to 0.0001 pg / mL.
The total amount of liquid feeding, that is, the volume of the flow path is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL.
The flow rate of liquid feeding is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.

Cleaning step The cleaning step is a step of cleaning at least one of the surface of the plasmon excitation sensor obtained through the step (a) and the surface of the plasmon excitation sensor obtained through the step (b) described later. . This washing step may be included in the assay method according to the present invention.

  However, in the assay method according to the present invention, chemiluminescence from the chemiluminescent molecule 22 is present in the vicinity of the plasmon excitation sensor 10 due to the molecular lifetime of the active molecule 41 generated from the photosensitizing layer 113. Only occurs when Therefore, the chemiluminescence from the chemiluminescent molecule 22 is substantially limited to the luminescence from the chemiluminescent molecule 22 immobilized on the plasmon excitation sensor 10, and from the chemiluminescent molecule-modified ligand complex 20 existing in the solution. Luminescence does not need to be considered in normal cases. From this, in the assay method according to the present invention, the washing step in step (b) is not an essential step. Rather, the washing step in step (b) may not be included so that real-time measurement can be performed. preferable.

  As the washing solution used in the washing step, for example, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer solution used in the reaction of steps (a) and (b), What contains 00001-1 weight% is desirable.

The temperature and flow rate at which the cleaning liquid is circulated are preferably the same as the “temperature and flow rate at which the liquid feed is circulated” in step (a).
The time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

<Step (b)>
In the detection method of the present invention, in the step (b), the chemiluminescent molecule-modified ligand complex comprising the second ligand 21 and the chemiluminescent molecule 22 is added to the specimen-immobilized plasmon excitation sensor obtained in the above step (a). 20 is a step in which a chemiluminescent molecule-fixed plasmon excitation sensor is obtained by contacting 20 and reacting them. Referring to FIG. 1, in this chemiluminescent molecule-fixed plasmon excitation sensor, the chemiluminescent molecule-modified ligand complex 20 is bound to the plasmon excitation sensor 10 via an analyte 31. This makes it possible to quantify the amount of analyte-ligand complex produced in the form of chemiluminescence in step (c) described below.

<Chemiluminescent molecule-modified ligand complex>
The chemiluminescent molecule-modified ligand complex 20 used in the present invention includes a second ligand 21 and chemiluminescent molecules 22.

Second Ligand In the assay method of the present invention, the second ligand 21 is a ligand used for the purpose of labeling the analyte 31 with the chemiluminescent molecule 22, and may be the same as the first ligand 12. , May be different. However, when the primary antibody used as the first ligand 12 is a polyclonal antibody, the secondary antibody used as the second ligand 21 may be a monoclonal antibody or a polyclonal antibody. When is a monoclonal antibody, the secondary antibody is preferably a monoclonal antibody that recognizes an epitope that the primary antibody does not recognize, or a polyclonal antibody.

Chemiluminescent molecule In the present invention, “chemiluminescent molecule” means a molecule that emits light when excited by a chemical reaction with an active molecule such as singlet oxygen in the present invention.

  The chemiluminescent molecule 22 used in the present invention is a molecule that generates chemiluminescence by reacting with the active molecule generated in the photosensitizing layer 113, and typically generates chemiluminescence in the presence of singlet oxygen. Is a molecule. The chemiluminescent molecule 22 is usually an electron-rich compound, and when in contact with singlet oxygen, a chemical reaction occurs between the chemiluminescent molecule 22 and the singlet oxygen to form an excited intermediate molecular species. Thereafter, when this intermediate molecular species further changes to a more stable molecule, fluorescence of a wavelength corresponding to the energy difference is generated. In the present invention, chemiluminescent molecules that emit light in the wavelength range of 500 to 620 nm are preferably used. In this way, by performing detection in a wavelength region that is farther shorter than the wavelength of incident light, the influence of noise on the chemiluminescent signal can also be suppressed. In addition, in such a wavelength region, it is possible to perform detection using various light receiving systems such as a CCD other than a photomultiplier tube. Examples of such molecules include compounds such as bis-indolylmaleimide, luminol, lucigeninl, Cypridina luciferin, 9-alkylidene acridans, enol ethers, enamines and 9-alkylidene xanthans.

Embodiment of Complex In the present invention, as long as the chemiluminescent molecule-modified ligand complex 20 is composed of the second ligand 21 and the chemiluminescent molecule 22, the binding mode is not particularly limited. However, from the viewpoint of enabling highly sensitive measurement by binding as many chemiluminescent molecules 22 as possible to the analyte 31 fixed on the plasmon excitation sensor 10, the chemiluminescent molecule-modified ligand complex 20 is: A fine particle modified with the chemiluminescent molecule 22 and the second ligand 21 is desirable.

  The fine particles (hereinafter referred to as “basic fine particles”) used as the material for such modified fine particles are fine particles that can be suspended without being precipitated in the liquid feeding. Examples of such basic fine particles include hydrophobic fine particles that are hydrophobic inside such as latex particles and capable of encapsulating a hydrophobic substance, and fine particles capable of encapsulating a hydrophilic substance in the inner aqueous layer such as liposome.

  Among these, the latex particles used in the present invention are granular polymer substances that can be suspended in water and insoluble in water, and can be obtained by a conventionally known method. In the present invention, the latex particles preferably have a particle size of 30 nm or more and 100 nm. The larger the particle size, the more chemiluminescent molecules 22 can be fixed and the luminescent signal is increased. However, if the particle size is too large, resistance may be easily received during liquid feeding.

  The latex particles used in the present invention can be obtained in a state in which the chemiluminescent molecules 22 are previously incorporated. For example, as in the method disclosed in Japanese Patent No. 4277479, a polymer obtained by radical polymerization of a vinyl monomer having a polymerizable ethylenically unsaturated double bond and the chemiluminescent molecule 22 are dissolved in an organic solvent. It can be produced as latex particles containing chemiluminescent molecules 22 by a method of (or dispersing) and removing the organic solvent after emulsification in water. As vinyl monomers having a polymerizable ethylenically unsaturated double bond used here, vinyl acetate, methyl acrylate, ethyl acrylate, n-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, acrylic acid Isononyl, dodecyl acrylate, octadecyl acrylate, 2-phenoxyethyl acrylate, methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, 2-ethylhexyl methacrylate, 2-hydroxyethyl methacrylate, dodecyl methacrylate , Octadecyl methacrylate, cyclohexyl methacrylate, stearyl methacrylate, benzyl methacrylate, glycidyl methacrylate, phenyl methacrylate, styrene, α-methylstyrene, acrylonitrile Acetoacetoxyethyl methacrylate, glycidyl methacrylate of soybean oil fatty acid modified products: include (Blenmer G-FA manufactured by NOF Corporation) and the like. Here, a monomer having an appropriate functional group such as an amino group or a carboxyl group is mixed with the vinyl monomer having a polymerizable ethylenically unsaturated double bond and used in the latex particles, so that Functional groups used for immobilization can be introduced.

  On the other hand, the liposome used in the present invention has an outer shell composed of an amphiphilic bilayer surrounding a certain volume of water or an aqueous solution, and usually has a particle size of 10 nm to 100 μm. The amphiphilic bilayer used here is usually composed of mixed lipid components such as phospholipids.

The method for producing such a liposome is not particularly limited, but from the viewpoint that the liposome surface can be efficiently modified, for example, a membrane emulsification method, a stirring emulsification method, a droplet method, a contact method pore membrane, etc. are used. In the two-stage emulsification method using the membrane emulsification method, a microchannel emulsification method and an emulsification method using a shirasu porous glass membrane (hereinafter, “SPG membrane”) are used. Preferably used. As a typical procedure,
(I) In the primary emulsification step, a W1 / O emulsion is formed by dispersing the first aqueous phase (W1) containing the mixed lipid component in an appropriate organic solvent (O) that is difficult to mix with water,
(Ii) In the subsequent secondary emulsification step, the W1 / O emulsion is dispersed in the second aqueous phase (W2) by using an appropriate emulsification method in the presence of the second mixed lipid component, whereby W1 / O / W2 Forming an emulsion,
(Iii) Thereafter, liposomes can be formed by removing the organic solvent (O) contained in the W1 / O / W2 emulsion. For example, when the membrane emulsification method is used, the W1 / O emulsion is dispersed in the second aqueous phase (W2) by using, for example, a microchannel or SPG membrane having a required pore size in the secondary emulsification step. Can do.

  The composition of the mixed lipid component used for the production of the liposome is not particularly limited, but in general, phospholipid (animal and plant derived lecithin; Glycerophospholipids, which are fatty acid esters; sphingophospholipids; derivatives thereof, and the like, and sterols (cholesterol, phytosterol, ergosterol, derivatives thereof, etc.) that contribute to the stabilization of lipid membranes. Lipids, glycols, aliphatic amines, long chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.), and other compounds that impart various functions may be blended. In the present invention, neutral phospholipids such as dipalmitoyl phosphatidylcholine (DPPC), dioleyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG) are commonly used as the phospholipid. The mixing ratio of the mixed lipid component may be appropriately adjusted according to the application while considering properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.

  In addition, the second mixed lipid component can be the same component as the mixed lipid component used in the primary emulsification step, and even if it has the same composition as the mixed lipid component used in the primary emulsification step, There may be. Here, in the present invention, the second ligand 21 and the chemiluminescent molecule 22 can be immobilized by including a lipid component having an appropriate functional group such as an amino group or a carboxyl group in the second mixed lipid component. It is preferable to introduce a functional group on the liposome surface. Examples of lipid components having such an appropriate functional group include distearoyl phosphatidylethanolamine (DSPE), and a functional group is introduced via a linker moiety such as a PEG group such as DSPE-PEG-COOH. It may be. In addition, a method is also known in which the liposome surface is coated with a polysaccharide such as pullulan and an antibody fragment is bound to a substituted aminoethylcarbamylmethyl group via γ-maleimidobutyloxysuccinimidyl.

In the secondary emulsification step, the second aqueous phase (W2) may contain a dispersing agent such as protein, polysaccharide, and nonionic surfactant.
For the basic particles such as latex particles and liposomes thus obtained, the second ligand 21 and the chemiluminescent molecule 22 can be immobilized using a conventionally known coupling method. For example, immobilization using conventional methods such as active ester method, amine coupling, coupling method based on reaction of iodoacetamide and thiol group, and method using affinity such as biotin-streptavidin bond Can do. It should be noted that only the second ligand 21 is fixed by the same method for fine particles into which the chemiluminescent molecules 22 are introduced when the basic fine particles are produced, such as latex particles containing the chemiluminescent molecules 22. You may make it.

  The concentration of the chemiluminescent molecule-modified ligand complex 20 produced in this manner during feeding is preferably 0.001 to 10,000 μg / mL, and more preferably 1 to 1,000 μg / mL.

<Step (c)>
In the detection method of the present invention, in step (c), a prism is applied to the chemiluminescent molecule-fixed plasmon excitation sensor obtained in step (b) from the surface of the transparent dielectric substrate 111 opposite to the metal thin film 112. In this step, the amount of fluorescence emitted from the excited chemiluminescent molecules 22 is measured by irradiating with laser light.

  Step (c) is usually performed by detecting fluorescence emission from the chemiluminescent molecule 22 bound to the chemiluminescent molecule-modified ligand complex 20 (sometimes referred to as “chemiluminescence” in the present invention). Specifically, the plasmon excitation sensor obtained by the step (b) is irradiated with laser light from the surface of the transparent dielectric substrate 111 opposite to the metal thin film 112 via a prism, and surface plasmon resonance The active molecule 41 is generated from the photosensitized layer 113 excited through, and the amount of fluorescence emitted from the chemiluminescent molecule 22 by the reaction with the active molecule 41 (in the present invention, sometimes referred to as “chemiluminescence amount”). ) Is measured.

  In the assay method of the present invention, chemiluminescence is generated by interposing active molecules 41 having a short molecular lifetime such as singlet oxygen, so that only the chemiluminescent molecules 22 fixed to the plasmon excitation sensor 10 are generated. Chemiluminescence, and it is not necessary to remove unreacted chemiluminescent molecule-modified ligand complex 20 by washing in the measurement in the step (c). Therefore, in the assay method of the present invention, it is possible to perform real-time measurement in which step (c) is performed simultaneously with step (b). The fact that real-time measurement is possible means that the amount of chemiluminescence at the end of the reaction can be estimated from the rate of increase in the amount of chemiluminescence from the start of the reaction, which is advantageous for short-time measurement. Is a point. In addition, since the SPFS-based assay method does not require a washing step between step (b) and step (c) that had to be performed to remove unreacted fluorescent dye and the like, measurement can be performed for a short time. There is an advantage that becomes possible.

Optical system The light source used in the present invention is not particularly limited as long as it can cause plasmon excitation in the metal thin film 112, but in terms of unity of wavelength distribution and intensity of light energy, It is preferable to use light as a light source. It is desirable to adjust the energy and photon amount immediately before the laser light enters the prism through the optical filter.

  Irradiation with laser light generates surface plasmons on the surface of the metal thin film 112 under the total reflection attenuation condition (ATR). Due to the electric field enhancement effect of the surface plasmon, the photosensitizer contained in the photosensitizing layer 113 is excited by photons increased to several tens to several hundred times the amount of irradiated photons, and active molecules 41 such as singlet oxygen are generated. Let Then, this active molecule 41 causes a chemical reaction with the chemiluminescent molecule 22 to form an intermediate molecular species in an excited state, and when this intermediate molecular species further changes to a reaction product in a more stable state, the energy difference The fluorescence of the wavelength corresponding to is produced. Note that the amount of photon increase due to the electric field enhancement effect depends on the refractive index of the transparent dielectric substrate 111 and the metal species and film thickness of the metal thin film 112, but is usually about 10 to 20 times that of gold.

  Examples of the “laser light” include an LD (laser diode) laser having a wavelength of 200 to 900 nm, 0.001 to 1,000 mW, a wavelength of 230 to 800 nm (the resonance wavelength is determined by the metal species used for the metal thin film 112), and 0. A semiconductor laser of 0.01 to 100 mW can be used.

  The “prism” is intended to allow the laser light through various filters to efficiently enter the plasmon excitation sensor, and preferably has the same refractive index as that of the transparent dielectric substrate 111. In the present invention, various prisms for which total reflection conditions can be set can be selected as appropriate, and therefore, there is no particular limitation on the angle and shape. Examples of such commercially available prisms include those similar to the above-mentioned commercially available “glass-made transparent flat substrate”. The prism may be incorporated in the transparent dielectric substrate 111 as a prism portion of the integrated structure block.

  Examples of the “optical filter” include a neutral density (ND) filter and a diaphragm lens. The “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.

The “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons.
“Cut filters” are external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattering light (excitation light originated from plasmon A filter that removes various types of noise light such as scattered light generated by the influence of structures or deposits on the surface of the excitation sensor), autofluorescence of the enzyme fluorescent substrate, and examples thereof include interference filters and color filters. It is done.

  The “collecting lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As a simple condensing system, a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

  As the “chemiluminescence detector”, a detection system usually used for SPFS detection can be used, and a photomultiplier tube (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) is preferable from the viewpoint of ultrahigh sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed. In the assay method according to the present invention, it is possible to select a detection wavelength such that the detected chemiluminescence wavelength is shorter than the wavelength of the normal incident light and is in the wavelength region near green. There is an advantage that it is easy to perform detection using a variety of light receiving systems.

  Moreover, in the assay method of the present invention, as in the assay method based on SPFS, the light source is located on the opposite side of the plasmon excitation sensor 10 with respect to the chemiluminescence detection unit, and measurement is performed under the total reflection condition. Therefore, the detection of chemiluminescence does not require special means for eliminating noise due to incident light, such as time-resolved measurement, as in a chemiluminescence assay system that does not use plasmon excitation such as the LOCI method.

Drive Device In the present invention, the optical system may be integrated in the form of a “drive device”. The drive apparatus of this invention is for implementing this invention using the said plasmon excitation sensor.

  The “driving device” includes at least a light source, various optical filters, a prism, a cut filter, a condenser lens, and a chemiluminescence detector. It is preferable to have a liquid feeding system combined with a plasmon excitation sensor when handling a sample liquid, a cleaning liquid, and the like. The type of liquid feeding system is not limited as long as the object of the present invention can be achieved. For example, a microchannel device connected to a liquid pump may be used.

  In addition, a surface plasmon resonance (SPR) detection unit, that is, a photodiode as a light receiving sensor dedicated to SPR, an angle variable unit for adjusting the optimum angle of SPR and SPFS (to determine total reflection attenuation (ATR) conditions with a servomotor) In addition, the angle of 30 to 85 ° can be changed by synchronizing the photodiode and the light source with a resolution of preferably 0.01 ° or more.), A computer for processing information input to the chemiluminescence detector May also be included.

Preferred embodiments of the light source, the optical filter, the cut filter, the condensing lens, and the chemiluminescence detector are the same as those described above.
As the “liquid feed pump”, for example, a micro pump suitable for a small amount of liquid feed, a syringe pump with high feed accuracy and low pulsation, which is preferable but cannot be circulated, a simple and excellent handleability but a small amount of liquid feed For example, a tube pump may be difficult.

<Step (d)>
In the detection method of the present invention, step (d) is a step of calculating the amount of analyte 31 contained in the specimen from the measurement result obtained in step (c).

  More specifically, a calibration curve is created by performing measurement with a target antigen or target antibody at a known concentration, and the target antigen amount or target antibody amount in the sample to be measured is measured based on the created calibration curve. This is a step of calculating from the signal.

Assay S / N ratio Further, in step (d), the “blank luminescence signal” measured before step (a), the “assay luminescence signal” obtained in step (c), and anything modified When the signal obtained by measuring SPFS while flowing ultrapure water while fixing a non-metal substrate in the flow path is “initial noise”, the assay S / N ratio represented by the following formula (1a) is Can be calculated:
Assay S / N ratio = | Ia / Io | / In (1a)
(In the above formula (1a), Ia is the assay luminescence signal, Io is the blank luminescence signal, and In is the initial noise).

However, in calculating the assay S / N ratio, in practice, instead of the above formula (1a), the “assay noise signal” in the case where the concentration of the analyte 31 contained in the sample is 0 is used as a reference. It may be calculated according to equation (1b):
Assay S / N ratio = | Ia | / | Ian | (1b)
(In the above formula (1b), Ian is the assay noise signal and Ia is the assay luminescence signal as in the case of the above formula (1a)).

〔kit〕
The kit of the present invention is characterized by being used in the assay method of the present invention. In carrying out the assay method of the present invention, the first ligand 12, the chemiluminescent molecule-modified ligand complex 20, and the sample are used. Except for the above, for example, a primary antibody, a ligand such as an antigen (that is, the analyte 31 contained in the sample is not limited to an antigen, and may be an antibody) and a sample. It is preferred to include everything needed except for and secondary antibodies.

  For example, by using the kit of the present invention, blood or serum as a specimen, and an antibody against a specific tumor marker, the content of the specific tumor marker can be detected with high sensitivity and high accuracy. From this result, the presence of a preclinical noninvasive cancer (carcinoma in situ) that cannot be detected by palpation or the like can be predicted with high accuracy.

  Specifically, the plasmon excitation sensor chip 20; a solution or dilution liquid for dissolving or diluting the specimen; a solution used in the reaction of the plasmon excitation sensor chip 20 and the specimen; and a cleaning reagent, Various equipment or materials required for carrying out the assay method of the present invention and the above-mentioned “drive device” can also be included.

  Further, the kit element may include a standard material for preparing a calibration curve, instructions, a necessary set of equipment such as a microtiter plate capable of simultaneously processing a large number of samples, and the like.

[Method for adjusting photosensitizer]
(R-1: Adjustment of photosensitizing layer coating solution)
A round bottom flask is equipped with a hot water bath, 500 g of ethyl acetate is heated to reflux, and 20 g of stearyl methacrylate, 50 g of methyl methacrylate, 30 g of 2-acetoacetoxyethyl methacrylate, and 0.1 g of N, N'-azobisisovaleronitrile are added. The dissolved mixed solution was added dropwise over 2 hours and reacted at the same temperature for 10 hours to obtain an ethyl acetate solution of the polymer.
5 ml of this resin solution was weighed, and further 1 mg of copper phthalocyanine was added and stirred to prepare a photosensitizer coating solution R-1.

(R-2: Adjustment of photosensitizing layer coating solution)
5 ml of a solution prepared by dissolving 100 g of polyvinyl butyral BL-S (manufactured by Sekisui Chemical Co., Ltd.) in 500 ml of ethyl acetate was weighed, and 1 mg of copper phthalocyanine was further added and stirred to prepare a photosensitizer coating solution R-2.

[Method for preparing labeled antibody fine particles]
<Polymer synthesis>
A dropping device, a thermometer, a nitrogen gas introduction tube, a stirring device, and a reflux condenser were attached to a 3 liter four-necked flask, and 1000 g of ethyl acetate was heated to reflux. Further, a mixture of 60 g of styrene, 10 g of lauryl methacrylate, 10 g of stearyl methacrylate, 10 g of acetoacetoxyethyl methacrylate, 10 g of methacrylic acid, and 4 g of N, N′-azobisisovaleronitrile was added dropwise over 2 hours. After reacting at 70 ° C. for 5 hours, a polymer solution was obtained.

<Manufacture of luminescent compound fine particles>
In a pot of Claremix CLM-0.8S (M Technic Co., Ltd.), 15 g of luminol, 20 g of the polymer solution, 10 g of polyvinyl butyral BL-S (Sekisui Chemical Co., Ltd.) and 140 g of ethyl acetate were added and stirred. . 15 g of Aqualon KH-05 (Daiichi Kogyo Seiyaku Co., Ltd.) was added to 245 g of pure water, added to the dye solution, and then emulsified for 5 minutes at a rotational speed of 20000 rpm. Then, ethyl acetate was removed under reduced pressure to obtain a dispersion of luminescent compound fine particles.

<Adjustment of luminescence labeled antibody>
10 mg of the luminescent compound fine particles were weighed and dissolved in 1 mL of 25 mM MES (2-morpholinoethanesulfonic acid) buffer (pH 6.0), and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC :( The carboxyl group of the luminescent compound fine particles was activated with 50 mM, manufactured by Doujin Chemical Laboratory, and 50 mM N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific). 1.4 mg / mL anti-α-fetoprotein (AFP) monoclonal antibody (6D2, manufactured by Mikuli Immuno Laboratory Co., Ltd.) was added to 100 uL (10 equivalents) and reacted at room temperature for 1 hour to react with microparticles. After the reaction, the mixture was purified by centrifugal ultrafiltration (Millipore) to obtain a luminescent compound microparticle-labeled anti-AFP monoclonal antibody solution. The obtained antibody solution was quantified for protein and fluorescent dye concentrations with an absorbance meter and stored at 4 ° C.

[Method for preparing comparatively labeled antibody]
(Labeled secondary antibody 1: Preparation of “Alexa Fluor 647” labeled anti-AFP monoclonal antibody)
An anti-α fetoprotein (AFP) monoclonal antibody (6D2, 2.5 mg / mL, manufactured by Mikuli Immuno Laboratory Co., Ltd.) was prepared using a commercially available Alexa Fluor647 labeling kit (Molecular Probes), and Alexa Fluor 647-labeled anti-AFP was prepared. A monoclonal antibody solution (Comparative labeled antibody 1) was obtained. The obtained antibody solution was quantified for protein and fluorescent dye concentrations with an absorbance meter and stored at 4 ° C.

[ Reference Example 1] Sensor substrate containing photosensitizer in dextran layer (Step 1: Formation of metal film)
A glass transparent flat substrate “S-LAL 10” (manufactured by OHARA INC., Refractive index [n _d ] = 1.72) having a thickness of 1 mm was plasma cleaned with a plasma dry cleaner. A gold film was formed on the plasma cleaned substrate surface by sputtering. The thickness of the gold film was 48 nm.

(Step 2: Formation of immobilized film)
The transparent substrate obtained in the step 1 is immersed in an ethanol solution containing 1 mM of 10-amino-1-decanthiol for 12 hours or more, and SAM (Self Assembled Monolayer; Molecular film) was formed. The substrate was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun. The obtained substrate was immersed in a 1 mg / mL aqueous solution of carboxymethyl dextran (manufactured by Meito Sangyo Co., Ltd., molecular weight 5 million, substitution degree 1.08). Furthermore, 0.5 mM each of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Dojin Chemical Laboratories) and N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) The mixture was allowed to react for 1.5 hours at room temperature, and amide coupling between the amino group terminal of SAM and the carboxyl group of carboxymethyldextran was performed to immobilize carboxymethyldextran. After completion of the reaction, 1N sodium hydroxide aqueous solution was dropped onto the substrate surface and reacted at room temperature for 30 minutes to convert the activated carboxyl group into carboxylic acid. In this manner, a plasmon excitation sensor chip having carboxymethyldextran immobilized on the surface was obtained.

(Step 3: Immobilization of primary antibody)
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Doujin Chemical Laboratory), N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) 25 mM MES containing 100 mM each ( (2-morpholinoethanesulfonic acid) buffer, 10 mM NaCl (pH 6.0) mixed solution is dropped on the plasmon excitation sensor chip obtained in the above-mentioned step 2, reacted at room temperature for 20 minutes, and incorporated in the sensor chip. Carboxymethyldextran was active esterified.

  Subsequently, 3,7-bis (aminoethylamino) phenothiazinium chloride (photosensitizer) 0.1 mM (25 mM MES buffer, 10 mM NaCl (pH 6.0)) was added dropwise and reacted at room temperature for 30 minutes. The photosensitizer was immobilized on carboxymethyl dextran.

Thereafter, an anti-α fetoprotein (AFP) monoclonal antibody (1D5, 1.8 mg / mL, manufactured by Mikuli Immuno Laboratory Co., Ltd.) (first ligand) was added to 25 mM MES (2- A solution obtained by diluting with morpholinoethanesulfonic acid (buffer) and 10 mM NaCl (pH 6.0) is dropped onto the substrate and reacted at room temperature for 30 minutes to link the first ligand to the carboxymethyldextran. did. After completion of the reaction, 1 N sodium hydroxide was dropped onto the surface of the substrate and reacted at room temperature for 30 minutes to convert the carboxyl group that had been active esterified into a carboxylic acid.
Finally, a 1% BSA-TBS buffer (pH 7.4) solution was dropped onto the substrate and subjected to a blocking treatment by reacting at room temperature for 40 minutes to obtain a plasmon excitation sensor.

(Step 4: Construction of flow path)
A flow made of polymethyl methacrylate (PMMA) having a flow path length of 7 mm and a width of 2 mm and a thickness of 2 mm for forming a measurement region on the surface of the plasmon excitation sensor obtained in step 3 on which the antibody is bound. I put the road. In this PMMA channel, a hole for introducing a liquid feed (liquid feed inlet) and a hole for discharging a liquid (liquid feed outlet) are formed. A laminate of these sensor substrates and PMMA flow paths was crimped at the outer periphery and fixed with screws to construct a plasmon excitation sensor.

  A silicone rubber tube and a peristaltic pump were connected to the liquid feeding inlet and the liquid feeding outlet of the plasmon excitation sensor section (hereinafter, unless otherwise specified, all the liquid feeding and circulation of various fluids are performed by such a tube and peristaltic pump). Was performed).

(Step 5: Signal measurement)
First, a solution obtained by diluting the plasmon excitation sensor manufactured by the above steps 1 to 4 with PBS buffer (pH 7.4) so that AFP (2.0 mg / mL solution, Acris Antibodies GmbH) is 0.1 ng / mL. Was allowed to flow at 5000 μL / min for 25 minutes.

  Subsequently, the luminescent compound fine particle-labeled secondary antibody anti-AFP monoclonal antibody (fluorescent dye precursor-modified ligand complex) prepared as described above was adjusted to 10 μg / mL with 1% BSA-PBS buffer (pH 7.4). The diluted solution was allowed to flow at 5000 μL / min for 5 minutes to obtain a plasmon excitation sensor on which the second ligand was immobilized.

  The emission signal of the plasmon excitation sensor thus obtained was measured using an SPFS measurement apparatus. Laser light (640 nm, 40 μW) was irradiated from the back side of the surface plasmon excitation sensor to which the second ligand was fixed via a prism, and the amount of fluorescence emitted from the sensor surface was measured with a CCD. This measured value was defined as “assay luminescence signal”.

On the other hand, with respect to another plasmon excitation sensor manufactured in the same manner as in Steps 1 to 4 of Reference Example 1, AFP (2.0 mg / mL solution, Acris Antibodies GmbH) was 0.1 ng in the first step of Step 5 above. 1% BSA-PBS buffer (pH 7.4) containing no AFP (0 ng / mL) instead of a solution diluted with PBS buffer (pH 7.4) so as to be equal to / mL. The amount of fluorescence was measured by the procedure, and the measured value was defined as “assay noise signal”.

[Example 2]
In Reference Example 1, a plasmon excitation sensor chip and a plasmon excitation sensor were produced in the same manner as in Reference Example 1 except that Steps 1 to 3 were changed as described below, and assay luminescence signals and assay noise signals were measured.

(Step 1: Formation of metal film)
A glass transparent flat substrate “S-LAL 10” (manufactured by OHARA INC., Refractive index [n _d ] = 1.72) having a thickness of 1 mm was plasma cleaned with a plasma dry cleaner. A gold film was formed on the plasma cleaned substrate surface by sputtering. The thickness of the gold film was 50 nm. Further, titanium oxide was deposited by direct sputtering to form a titanium oxide layer having a thickness of 10 nm on the gold film surface.

(Step 2: Formation of photosensitizing layer)
The above-mentioned photosensitizing layer coating solution R-1 was applied to the transparent substrate obtained in the above step 1 using a spin coater to form a photosensitizing layer having a dry film thickness of 10 nm. A 0.1N sodium hydroxide aqueous solution was dropped on the surface of the photosensitized layer, and the ester bond on the outermost surface of the photosensitized layer was hydrolyzed to form a carboxyl group. Further, 5 ml of an ethyl acetate solution in which 0.02 g of 1,4-diaminobutane and 0.1 g of dicyclohexylcarbodiimide were dissolved was allowed to act to introduce amino groups onto the substrate surface. The substrate was immersed in a 1 mg / mL aqueous solution of carboxymethyl dextran (manufactured by Meito Sangyo Co., Ltd., molecular weight 5 million, substitution degree 1.08). Furthermore, 0.5 mM each of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Dojin Chemical Laboratories) and N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) The mixture was allowed to react for 1.5 hours at room temperature, and amide coupling between the SAM at the amino group terminal and the carboxyl group of dextran was carried out to immobilize carboxymethyldextran. After completion of the reaction, 1N sodium hydroxide aqueous solution was dropped onto the substrate surface and reacted at room temperature for 30 minutes to convert the activated carboxyl group into carboxylic acid. In this manner, a plasmon excitation sensor chip having carboxymethyldextran immobilized on the surface was obtained.

(Step 3: Immobilization of primary antibody)
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Doujin Chemical Laboratory), N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) 25 mM MES containing 100 mM each ( (2-morpholinoethanesulfonic acid) buffer, 10 mM NaCl (pH 6.0) mixed solution is dropped on the plasmon excitation sensor chip obtained in the above-mentioned step 2, reacted at room temperature for 20 minutes, and incorporated in the sensor chip. Carboxymethyldextran was active esterified.

Subsequently, an anti-α fetoprotein (AFP) monoclonal antibody (1D5, 1.8 mg / mL, manufactured by Mikuli Immuno Laboratory Co., Ltd.) (first ligand) was added to 25 mM MES (2 -Morpholinoethanesulfonic acid) A solution obtained by diluting with a buffer, 10 mM NaCl (pH 6.0) was dropped onto the substrate, reacted at room temperature for 30 minutes, and the first ligand was added to the carboxymethyldextran. Connected.
Finally, a 1% BSA-TBS buffer (pH 7.4) solution was dropped onto the substrate and subjected to a blocking treatment by reacting at room temperature for 40 minutes to obtain a plasmon excitation sensor.

[Example 3]
In Example 2, the photosensitizing layer coating solution R-2 was used instead of the photosensitizing layer coating solution R-1 as the photosensitizing layer coating solution used for forming the photosensitizing layer in Step 2. Except for this, a surface plasmon excitation sensor was produced in the same manner as in Example 2, and assay luminescence signal and assay noise signal were measured.

[Comparative Example 1]
In Reference Example 1, a plasmon excitation sensor was produced in the same manner as in Reference Example 1 except that Steps 3 to 5 were changed as follows, and assay luminescence signal and assay noise signal were measured.

(Step 3: Immobilization of primary antibody)
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC: manufactured by Doujin Chemical Laboratory), N-hydroxysuccinimide (NHS: manufactured by Thermo Scientific) 25 mM MES containing 100 mM each ( 2-Morpholinoethanesulfonic acid) buffer, 10 mM NaCl (pH 6.0) mixed solution is dropped onto the plasmon excitation sensor chip obtained in Step 2 and reacted at room temperature for 20 minutes, and the carboxy incorporated in the sensor chip. Methyl dextran was active esterified.

  Subsequently, an anti-α fetoprotein (AFP) monoclonal antibody (1D5, 1.8 mg / mL, manufactured by Mikuli Immuno Laboratory Co., Ltd.) (first ligand) was added to 25 mM MES (2 -Morpholinoethanesulfonic acid) A solution obtained by diluting with a buffer, 10 mM NaCl (pH 6.0) was dropped onto the substrate, reacted at room temperature for 30 minutes, and the first ligand was added to the carboxymethyldextran. Connected.

  Finally, a 1% BSA-TBS buffer (pH 7.4) solution was dropped onto the substrate, and a blocking treatment was performed by reacting at room temperature for 40 minutes to obtain a surface plasmon excitation sensor.

(Step 4: Construction of flow path)
A flow made of polymethyl methacrylate (PMMA) having a flow path length of 7 mm and a width of 2 mm and a thickness of 2 mm for forming a measurement region on the surface of the plasmon excitation sensor obtained in step 3 on which the antibody is bound. I put the road. In this PMMA channel, a hole for introducing a liquid feed (liquid feed inlet) and a hole for discharging a liquid (liquid feed outlet) are formed. A laminate of these sensor substrates and PMMA flow paths was crimped at the outer periphery and fixed with screws to construct a plasmon excitation sensor.

  A silicone rubber tube and a peristaltic pump were connected to the liquid feeding inlet and the liquid feeding outlet of the plasmon excitation sensor section (hereinafter, unless otherwise specified, all the liquid feeding and circulation of various fluids are performed by such a tube and peristaltic pump). Was performed).

(Step 5: Signal measurement)
First, a solution obtained by diluting the plasmon excitation sensor manufactured by the above steps 1 to 4 with PBS buffer (pH 7.4) so that AFP (2.0 mg / mL solution, Acris Antibodies GmbH) is 0.1 ng / mL. Was allowed to flow at 5000 μL / min for 25 minutes. As a washing step, a TBS solution (pH 7.4) containing 0.05% Tween 20 was allowed to flow at 5000 μL / min for 2 minutes.

  Subsequently, labeled secondary antibody 1 prepared as described above 1: 1% BSA-PBS buffer (pH 7. 5) so that the “Alexa Fluor 647” labeled anti-AFP monoclonal antibody (secondary ligand) is 2.5 μg / mL. The solution diluted in 4) was allowed to flow at 5000 μL / min for 5 minutes. As a washing step, a TBS solution (pH 7.4) containing 0.05% Tween 20 was flowed at 5000 μL / min for 2 minutes to obtain a plasmon excitation sensor in which the second ligand was immobilized.

  The emission signal of the plasmon excitation sensor thus obtained was measured using an SPFS measurement apparatus. Laser light (640 nm, 40 μW) was irradiated from the back side of the plasmon excitation sensor on which the second ligand was fixed via a prism, and the amount of fluorescence emitted from the sensor surface was measured with a CCD. This measured value was defined as “assay luminescence signal”.

  On the other hand, with respect to another surface plasmon excitation sensor manufactured in the same manner as in Steps 1 to 4 of Comparative Example 1, AFP (2.0 mg / mL solution, Acris Antibodies GmbH) is 0. 1% BSA-PBS buffer (pH 7.4) containing no AFP (0 ng / mL) was used instead of the solution diluted with PBS buffer (pH 7.4) to 1 ng / mL. The amount of fluorescence was measured by the same procedure, and the measured value was defined as “assay noise signal”.

[Comparative Example 2]
A plasmon excitation sensor chip and a plasmon excitation sensor were produced in the same manner as in Comparative Example 1 except that Step 5 was changed as follows, and assay luminescence signal and assay noise signal were measured. In Comparative Example 2, a short-time treatment was attempted without washing after the sandwich assay.

(Step 5: Signal measurement)
First, a solution obtained by diluting the plasmon excitation sensor manufactured by the above steps 1 to 4 with PBS buffer (pH 7.4) so that AFP (2.0 mg / mL solution, Acris Antibodies GmbH) is 0.1 ng / mL. Was allowed to flow at 5000 μL / min for 25 minutes.

  Subsequently, labeled secondary antibody 1 prepared as described above 1: 1% BSA-PBS buffer (pH 7. 5) so that the “Alexa Fluor 647” labeled anti-AFP monoclonal antibody (secondary ligand) is 2.5 μg / mL. The solution diluted in 4) was allowed to flow at 5000 μL / min for 5 minutes.

  Thereafter, laser light (640 nm, 40 μW) was irradiated from the back side of the plasmon excitation sensor to which the second ligand was fixed via a prism, and the amount of fluorescence emitted from the sensor surface was measured with a CCD. This measured value was defined as “assay luminescence signal”.

On the other hand, with respect to another plasmon excitation sensor manufactured by the above steps 1 to 4, except that 1% BSA-PBS buffer (pH 7.4) containing no AFP (0 ng / mL) was flowed in the first step. The amount of fluorescence was measured by the same procedure as above, and the measured value was defined as “assay noise signal”.

For each of the above Examples and Comparative Examples, the S / N ratio was calculated from the assay signal and the blank signal according to the following formula.
S / N ratio = | (assay luminescence signal) | / | (assay noise signal) |

As is apparent from the results in Table 1, the assay luminescence signal was high during the assay of Comparative Example 1 and Comparative Example 2 (after 3 minutes) in which the conventional ordinary sandwich immunoassay was performed, but the assay noise signal was also high. This is because the fluorescence (nonspecific adsorption) of the labeled antibody in the flow path is also detected, and as a result, S / N cannot be obtained and a significant detection accuracy cannot be obtained. Significant detection accuracy cannot be obtained unless a washing step is performed after completion of the assay as in Comparative Example 1. On the other hand, in Reference Example 1 and Example 2 of the present invention, as can be seen from the assay noise signal, the fluorescent dye precursor and the developer are physically separated sufficiently during the assay. Fluorescence (nonspecific adsorption) is hardly detected. It can be seen that a signal corresponding to the supplemental amount of AFP is obtained and the S / N is also high. In addition, since the S / N is discriminated, even if the reaction is not performed until after the assay is completed, for example, in Examples 2 and 3 and Reference Example 1 , a significant S / N can be obtained even if the assay is performed for 3 minutes. N can be obtained, and it can be seen that AFP can be measured in a short time.

DESCRIPTION OF SYMBOLS 10 ... Plasmon excitation sensor 11 ... Plasmon excitation sensor chip 111 ... Transparent dielectric substrate 112 ... Metal thin film 113 ... Photosensitizing layer 12 ... 1st ligand 20 ... Chemistry Luminescent molecule-modifying ligand complex 21 ... second ligand 22 ... chemiluminescent molecule 31 ... analyte 41 ... active molecule

Claims (9)

A transparent dielectric substrate;
A metal thin film;
A layer containing a photosensitizer (photosensitizing layer) in this order ,
The photosensitizing layer comprises a singlet oxygen generator; and
A plasmon excitation sensor chip having a dielectric layer between the metal thin film and the photosensitizing layer .   The plasmon excitation sensor chip according to claim 1, wherein the photosensitizing layer further includes a dielectric. A plasmon excitation sensor in which a ligand is bonded to a surface of the plasmon excitation sensor chip according to any one of claims 1 and 2 on which the photosensitizing layer is present. The plasmon excitation sensor according to claim 3 , wherein the ligand is an antibody. An assay method comprising the following steps (a) to (d):
Step (a): a step of bringing a specimen into contact with the plasmon excitation sensor according to any one of claims 3 to 4 to obtain a specimen-fixed plasmon excitation sensor;
Step (b): A chemiluminescent molecule-immobilized plasmon excitation sensor is obtained by bringing a chemiluminescent molecule-modified ligand complex comprising a second ligand and a chemiluminescent molecule into contact with the analyte-immobilized plasmon excitation sensor and reacting them. Process;
Step (c): Excitation of the chemiluminescence excited by irradiating the chemiluminescent molecule-fixed plasmon excitation sensor with a laser beam from a surface of the transparent dielectric substrate opposite to the metal thin film via a prism. Measuring the amount of fluorescence emitted from the molecule; and
Step (d): A step of calculating the amount of the analyte contained in the sample from the measurement result obtained in the step (c). The assay method according to claim 5 , wherein the chemiluminescent molecule is a molecule that emits chemiluminescence in the presence of singlet oxygen. The assay method according to any one of claims 5 to 6 , wherein the chemiluminescent molecule-modified ligand complex is a fine particle modified with the chemiluminescent molecule and the second ligand. The assay method according to any one of claims 5 to 7 , wherein the specimen is at least one body fluid selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva. Plasmon excitation sensor chip according to any one of the claims 1-2;
A lysate or diluent for lysing or diluting the specimen;
A solution used in the reaction between the plasmon excitation sensor chip and the specimen; and
Kit containing cleaning reagents.