JP5835335B2 - Method for quantitative measurement of specific analytes using surface plasmon resonance and surface plasmon excitation enhanced fluorescence spectroscopy - Google Patents

Description

  The present invention relates to a specific analog using the principle of surface plasmon resonance (SPR) phenomenon and surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) applying the SPR phenomenon. The present invention relates to a method for quantitative measurement of light.

  Conventionally, proteins in blood, urine, and tissues have been widely used in disease diagnosis. Most proteins in the living body exist on the surface of proteins consisting of amino acids with sugar chains modified.

  Even for proteins with the same amino acid sequence (proteins with the same name), there are a wide variety of modified sugar chains, and it is known that the sugar chain structure differs depending on the state of the cell producing the protein.

  The relationship between such sugar chain changes and diseases has also been clarified, for example, α-fetoprotein (AFP) sugar chain fraction measurement as disclosed in Non-Patent Document 1, Patent Document 1, It is applied to carcinoembryonic antigen (CEA) sugar chain fraction measurement as disclosed in Non-Patent Documents 2 and 3.

  Among the AFP sugar chain fraction measurements, the AFP-L3 fraction that detects the change in sugar chain associated with canceration and binding to lectin (LCA) is used as a marker for liver cancer. AFP is divided into three parts, LCA non-binding fraction (L1), LCA weak binding fraction (L2), and LCA binding fraction (L3). L1 is mainly used in chronic hepatitis and cirrhosis, and L3 is used in liver cancer. Will increase.

  Therefore, by measuring the amount of L3 fraction in the total amount of AFP, it can be applied to discovery of liver cancer, discrimination between hepatitis and liver cancer, and the like.

  CEA sugar chain fraction measurement is mainly an index for adenocarcinoma, and is used for the discovery of colon cancer, stomach cancer, lung cancer, ovarian cancer, uterine cancer, and the like.

  In addition, cancerous changes in sugar chains are known in choriocarcinoma, rectal cancer, etc., and it has been found that measuring such cancerous changes in sugar chains is very useful for cancer diagnosis. It was.

  As a method for quantifying the proportion of glycoproteins in which sugar chains have changed, lectin column chromatography is used to separate them by columns using the difference in sugar chains that modify glycoproteins. By performing ELISA (Enzyme-Linked ImmunoSorbent Assay) measurement, a protein having a specific sugar chain can be quantified.

JP 61-292062 A

Sugar Chains of Human Cord Serum α-Fetoprotein: Characteristics of N-linked Sugar Chains of Glycoproteins Produced in Human Liver and Hepatocellular Carcinomas, K. Yamashita et al., Cancer Res., 53, 1 (1993) Carbohydrate Structures of Nonspecific Cross-reacting Antigen-2, a Glycoprotein Purified from Meconium as an Antigen Cross-reacting with Anticarcinoembryonic Antigen Antibody, K. Yamashita et al., Biol. Chem., 264, 17873 (1989) Structural Studies of the Carbohydrate Moieties of Carcinoembryonic Antigens, K. Yamashita et al., Cancer Res., 47, 3451 (1987)

  However, such a method includes a step of separating and purifying the antigen using a column, which causes problems such as a long time for measurement and loss of the antigen at the stage of column processing. In addition, a very large amount of lectin necessary for separating the antigen is consumed, which increases the measurement cost.

  In addition, the lectin used for fraction quantification has a low affinity and can be used only for quantification of a certain amount of antigen when it is used as a detection probe. When the amount of antigen is small, measurement such as ELISA is possible. The system could not detect and quantify well.

  By the way, in the case of detecting extremely small substances, as a specimen detection device that enables detection of such substances by applying physical phenomena of electrons, electrons and light are detected in a minute region such as the nanometer level. Applying the phenomenon of resonance (Surface Plasmon Resonance (SPR) phenomenon), for example, a surface plasmon resonance device (hereinafter referred to as “SPR device”) that detects minute analytes in a living body. It has been known.

  In addition, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) using the surface plasmon resonance (SPR) phenomenon, it is possible to perform analyte detection with higher accuracy than the SPR device. The surface plasmon excitation enhanced fluorescence spectrometer (hereinafter referred to as “SPFS device”) is also known as such an analyte detection device.

  In this surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the surface plasmon light is applied to the surface of the metal thin film under the condition that incident light such as laser light emitted from the light source is attenuated by total reflection (ATR) on the surface of the metal thin film. By generating (dense wave), the amount of photons included in the incident light irradiated from the light source is increased to several tens to several hundreds times, and the electric field enhancement effect of the surface plasmon light is obtained.

  This electric field enhancement effect efficiently excites the fluorescent substance bound (labeled) with the analyte trapped in the vicinity of the metal thin film, for example, a photon counting type photomultiplier tube (PMT) or CCD (Charge Coupled). By observing this fluorescence using a light detection means such as a Device) camera, it is possible to detect an extremely small amount of analyte at a very low concentration.

  In the present invention, by measuring the canceration change of a sugar chain using such an SPR device and an SPFS device, it can be quickly measured without using a column, and there is no loss of an antigen accompanying column processing. Another object of the present invention is to provide a method for quantitative measurement of a specific analyte that can suppress the consumption of a labeled probe such as a lectin.

The present invention was invented in order to achieve the problems and objects in the prior art as described above, and the specific analyte quantitative measurement method of the present invention includes:
A sensor chip having a dielectric member, a metal film formed on the dielectric member, and a ligand that captures an analyte including the specific analyte formed on the metal film is provided on the sensor chip. Providing a specimen containing light and contacting the ligand;
The metal film of the sensor chip is irradiated with incident light through the dielectric member, and the metal film reflected light reflected by the metal film is received by a light receiving unit, thereby receiving the metal. Measuring the amount of film reflected light; and
After measuring the amount of light reflected from the metal film, a step of reacting a fluorescently labeled probe for fluorescently labeling the specific analyte with the specific analyte;
Fluorescence generated by exciting the fluorescently labeled probe bound to the specific analyte with surface plasmon light generated by irradiating incident light through the dielectric member to the metal film of the sensor chip Measuring the amount of the fluorescence by receiving the light by the light detection means,
Calculating the total analyte concentration in the specimen based on the amount of light reflected by the metal film;
Calculating a specific analyte concentration in the specimen based on the amount of fluorescence;
Calculating a ratio of a specific analyte amount to a total analyte amount from the total analyte concentration and the specific analyte concentration;
It is characterized by having.

  By comprising in this way, it becomes unnecessary to isolate | separate and refine | purify a specific analyte beforehand by a column, and it can measure rapidly. Further, since column processing is not performed, problems such as loss of a specific analyte do not occur. Moreover, since the consumption of lectin can also be suppressed, measurement cost can also be reduced.

  Furthermore, since SPFS measurement is performed in order to quantify a specific analyte, even if the amount of the specific analyte is very small, the specific analyte can be detected with high accuracy. The ratio of the specific amount of analyte to the amount of light can be accurately measured.

  The quantitative measurement method of the present invention is characterized in that a channel is formed on the metal film, and the ligand is formed in a part of the channel.

  In this way, the sample liquid can be sent by forming the flow channel on the metal film, and the analyte in the sample liquid is reliably captured by the capturing body formed in a part of the flow channel. be able to.

  In the quantitative measurement method of the present invention, the ligand is a protein or a nucleic acid.

  The quantitative measurement method of the present invention is characterized in that the specific analyte has a sugar chain.

  In the quantitative measurement method of the present invention, the fluorescently labeled probe is a lectin that is labeled with a fluorescent dye and binds to the specific analyte.

  Thus, it can be used for quantitative measurement of α-fetoprotein (AFP), carcinoembryonic antigen (CEA), etc. For example, it can be used to detect AFP-L3 for accurate and rapid diagnosis of liver cancer. It can be carried out.

Further, in the quantitative measurement method of the present invention, the step of calculating the total analyte concentration in the specimen includes a calibration curve relating to the total analyte concentration and the amount of reflected light from the metal film prepared in advance from a plurality of standard antigens, While performing by calculating based on the light amount of the metal film reflected light measured by the step of measuring the light amount of the metal film reflected light,
The step of calculating a specific analyte concentration in the specimen is measured by a calibration curve relating to a specific analyte concentration and a fluorescence light amount prepared in advance from a plurality of standard antigens, and a step of measuring the fluorescence light amount. The calculation is performed based on the amount of fluorescent light.

  In this way, by preparing a calibration curve relating to the light amount of the reflected light from the metal film and a calibration curve relating to the light amount of the fluorescence in advance, the total analyte concentration and the specific analyte concentration can be accurately and quickly determined by a quantitative calculation means. Quantification can be performed.

  According to the present invention, after measuring the total analyte concentration by SPR measurement, it is not necessary to fractionate the analyte by measuring the specific analyte concentration by SPFS measurement. The concentration of the analyte can be measured accurately and rapidly. For example, by using it for detection of AFP-L3, it can be applied to disease diagnosis of liver cancer.

FIG. 1 is a schematic view schematically showing an outline of a quantitative measurement apparatus for explaining a specific analyte quantitative measurement method of the present invention. FIG. 2 is a partially enlarged view of the quantitative measurement apparatus of FIG. FIG. 3 is an enlarged schematic view schematically showing the sensor unit 38 before feeding the standard antigen (analyte 46). FIG. 4 is an enlarged schematic view schematically showing the sensor unit 38 after feeding the standard antigen (analyte 46). FIG. 5 is a graph showing changes with time in the SPR signal value, which is the difference between the SPR signal of each calibration curve sample AFP antigen solution and the blank signal. FIG. 6 is a graph showing the relationship between the SPR signal value of each calibration curve sample AFP antigen solution and the AFP concentration of each calibration curve sample AFP antigen solution. FIG. 7 is an enlarged schematic view schematically showing the sensor unit 38 after feeding the fluorescently labeled probe 48. FIG. 8 is a graph showing the relationship between the SPFS signal value of each calibration curve sample AFP antigen solution and the AFP-L3 concentration of each calibration curve sample AFP antigen solution.

Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.
1. Configuration of Quantitative Measurement Device FIG. 1 is a schematic diagram schematically showing an outline of a quantitative measurement device for explaining a quantitative measurement method for an analyte of the present invention, and FIG. 2 is a partially enlarged view of the quantitative measurement device of FIG. .

  As shown in FIGS. 1 and 2, the quantitative measurement device 10 of the present invention includes a prism-shaped dielectric member 12 having a substantially trapezoidal vertical cross-sectional shape, and a metal formed on a horizontal upper surface 12 a of the dielectric member 12. A sensor chip 16 having a film 14 is provided, and the sensor chip 16 is loaded in a sensor chip loading unit 18 of the quantitative measurement apparatus 10.

  Further, as shown in FIG. 1, a light source 20 is disposed on one side surface 12 b below the dielectric member 12, and incident light 22 from the light source 20 is below the dielectric member 12. Then, the light enters the side surface 12 b of the dielectric member 12 and is irradiated through the dielectric member 12 toward the metal film 14 formed on the upper surface 12 a of the dielectric member 12.

The material of the dielectric member 12 is not particularly limited, but optically transparent, for example, various inorganic materials such as glass and ceramics, natural polymers, and synthetic polymers can be used. From the viewpoints of stability, production stability, and optical transparency, those containing silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) are preferred.

The dielectric member 12 has a refractive index at the d-line (wavelength 588 nm) (n _d) is preferably from 1.40 to 2.20, thickness, preferably 0.01 to 10 mm, more preferably 0.5-5 mm. The size (vertical x horizontal) of the dielectric member 12 is not particularly limited.

In addition, the dielectric member 12 made of glass is commercially available as “BK7” (refractive index (n _d ) 1.52) and “LaSFN9” (refractive index (n _d ) 1.85 manufactured by Shot Japan Co., Ltd. ), “K-PSFn3” (refractive index (n _d ) 1.84), “K-LaSFn17” (refractive index (n _d ) 1.88) and “K-LaSFn22” (manufactured by Sumita Optical Glass Co., Ltd.) Refractive index (n _d ) 1.90) and “S-LAL10” (refractive index (n _d ) 1.72) manufactured by OHARA INC. Are preferable from the viewpoints of optical properties and detergency.

  The dielectric member 12 is preferably cleaned with acid and / or plasma before the metal film 14 is formed on the upper surface 12a.

  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.

  In this embodiment, the prism-shaped dielectric member 12 having a substantially trapezoidal vertical cross-sectional shape is used. However, the vertical cross-sectional shape may be a triangle (so-called triangular prism), a semicircular shape, a semi-elliptical shape, etc. The shape of the body member 12 can be changed as appropriate.

  The material of the metal film 14 formed on the upper surface 12a of the dielectric member 12 is not particularly limited, but is preferably at least selected from the group consisting of gold, silver, aluminum, copper, and platinum. It is made of one kind of metal, more preferably made of gold, and may be made of an alloy of these metals.

  Such a metal is suitable for the metal film 14 because it is stable against oxidation and, as will be described later, the electric field enhancement by surface plasmon light is increased.

  The method for forming the metal film 14 is not particularly limited, and examples thereof include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. It is done. It is preferable to use a sputtering method or a vapor deposition method because it is easy to adjust the thin film formation conditions.

  Further, the thickness of the metal film 14 is not particularly limited, but preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 ˜500 nm and their alloys: preferably in the range of 5 to 500 nm.

  In addition, from the viewpoint of the electric field enhancement effect described later, more preferable thicknesses of the metal film 14 are: gold: 20 to 70 nm, silver: 20 to 70 nm, aluminum: 10 to 50 nm, copper: 20 to 70 nm, platinum: 20 to It is desirable that the range is 70 nm and alloys thereof: 10 to 70 nm.

  If the thickness of the metal film 14 is within the above range, surface plasmon light, which will be described later, is easily generated, which is preferable. In addition, as long as the metal film 14 has such a thickness, the size (length × width) dimensions and shape are not particularly limited.

  Further, the light source 20 is not particularly limited as long as it can generate plasmon light on the metal film 14, but in terms of unity of wavelength distribution and intensity of light energy, Laser light such as a diode is preferably used as the light source. Laser light such as a photodiode desirably adjusts the energy and the amount of photons immediately before entering the prism through an optical filter.

  When laser light is used as the light source 20, for example, a laser diode (LD) with a wavelength of 200 to 900 nm, 0.001 to 1,000 mW; a semiconductor laser with a wavelength of 230 to 800 nm, 0.01 to 100 mW, or the like is used. Can be used.

  Examples of the optical filter include an ND (darkening) filter, a neutral density filter, and a diaphragm lens. The ND (darkening) filter, the neutral density filter, and the like are intended to adjust the amount of incident laser light. In particular, when a detector having a narrow dynamic range is used, it is preferable to use such an optical filter in order to perform highly accurate measurement.

  Further, a polarizing filter for converting the laser light emitted from the light source 20 into P-polarized light that efficiently generates surface plasmons on the metal film 14 can be provided between the light source 20 and the dielectric member 12. .

  Further, on the other side surface 12c below the dielectric member 12, as shown in FIG. 1, a light receiving means 26 for receiving the metal film reflected light 24 in which the incident light 22 is reflected by the metal film 14 is provided. ing.

  The light source 20 is provided with incident angle adjusting means (not shown) that can appropriately change the incident angle α1 of the incident light 22 irradiated from the light source 20 with respect to the metal film 14. On the other hand, the light receiving means 26 is also provided with a movable means (not shown), and even when the reflection angle of the metal film reflected light 24 changes, the metal film reflected light 24 is reliably received in synchronization with the light source 20. Is configured to do.

  Further, the incident angle adjusting means of the light source 20 and the moving means of the light receiving means 26 are not particularly limited. For example, the light source 20 and the light receiving means 26 are rotated (for example, using a stepping motor, a gear train, etc.). In FIG. 1, it can be configured to rotate around the irradiation position of the incident light on the metal film 14 in the plane.

  The sensor chip 16, the light source 20, and the light receiving means 26 constitute an SPR measurement unit 28 that performs SPR measurement of the quantitative measurement device 10 of the present invention.

  Above the sensor chip 16, there is provided a light detection means 32 for receiving the fluorescence 30 emitted from a fluorescent material as described later.

  Here, the light detecting means 32 is not particularly limited. For example, a photon counting type photomultiplier tube (PMT) or a CCD (Charge Coupled Device) image capable of multipoint measurement is used. A sensor, a complementary metal oxide semiconductor (CMOS) image sensor, or the like can be used.

  For example, a cut filter or a condenser lens can be provided between the sensor chip 16 and the light detection means 32.

  The cut filter includes external light (illumination light, ambient light, etc. outside the quantitative measurement apparatus 10), transmission component of incident light emitted from the light source 20, stray light (scattering component of incident light in various places), scattered light of plasmon light ( A filter that removes optical noise such as scattered light that originates from incident light and is generated due to the influence of structures or deposits on the surface of the sensor chip 16, and autofluorescence of a fluorescent material, for example, an interference filter, A color filter or the like can be used.

  The condensing lens is intended to efficiently condense the fluorescent signal generated by exciting the fluorescent substance onto the light detection means 32, and any condensing system can be used. As a simple condensing system, for example, a commercially available objective lens used in a microscope or the like, for example, manufactured by Nikon Corporation or manufactured by Olympus Corporation may be diverted. The magnification of the objective lens is preferably 10 to 100 times.

  The sensor chip 16, the light source 20, and the light detection means 32 constitute an SPFS measurement unit 34 that performs SPFS measurement of the quantitative measurement device 10 of the present invention.

  The light receiving means 26 and the light detecting means 32 are respectively connected to the quantitative calculation means 40, and the light amount of the metal film reflected light 24 received by the light receiving means 26 and the light amount of the fluorescence 30 received by the light detecting means 32. Are transmitted to the quantitative calculation means 40.

  Further, in the sensor chip 16 of the present embodiment, the flow path 36 is formed on the upper surface 14 a of the metal film 14. A part of the flow path 36 is also referred to as a ligand that specifically binds to an analyte containing a specific analyte (hereinafter referred to as a “ligand that captures an analyte containing a specific analyte”. Is provided). A sensor unit 38 is provided.

  Here, the analyte is not particularly limited, but for example, proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), nucleic acids (single-stranded or double-stranded). DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside, nucleotide and their modified molecules), carbohydrate (oligosaccharide, polysaccharide, sugar chain, etc.), lipid, or these Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, or the like.

  In addition, the specific analyte is not particularly limited as long as it specifically binds to a fluorescently labeled probe as described later among the above-described analytes. It can be an analyte having a sugar chain to be bound.

  In addition, for example, by using a fluorescently labeled probe that specifically binds to a phosphorylated protein as a protein phosphorylated from a specific analyte, the quantitative measurement method of the present invention can be applied to cancers such as p53 and K-ras. It can also be used for distinguishing differences in phosphorylation levels of related proteins, measuring the proportion of K-ras proteins having specific mutations, and the like.

  The ligand is not particularly limited as long as it specifically binds to an analyte including a specific analyte, and may be a protein or a nucleic acid.

  In this embodiment, the analyte is combined with the sensor unit 38 by sending a sample liquid containing a specific analyte to the flow path 36, and, as will be described later, by the SPR measurement unit 28 and the SPFS measurement unit 34. The light amount of the metal film reflected light 24 and the light amount of the fluorescence 30 are measured.

  Note that a configuration may be adopted in which the sensor portion 38 having the ligand immobilized thereon is provided on the metal film 14 without providing the flow path 36. When configured in this manner, a sample liquid containing a specific analyte is added to the sensor unit 38 using a dropper or the like, for example, thereby capturing the analyte containing the specific analyte in the sensor unit 38. Can be made.

  Thus, when the sensor unit 38 is provided on the metal film 14, for example, a SAM (Self-Assembled Monolayer) is formed on the metal film 14, and the solid phase is formed on the SAM. For example, a layer may be provided, and a ligand may be immobilized on the solid phase layer.

  In addition, as a single molecule constituting such a SAM, usually a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Doto Chemical Laboratory, Sigma Aldrich Japan Co., Ltd.) Particularly preferably, 10-carboxy-1-decanethiol is used. Carboxyalkanethiol having 4 to 20 carbon atoms has properties such as little optical influence of SAM formed using it, that is, high transparency, low refractive index, and thin film thickness. Therefore, it is preferable.

  A method for forming such a SAM is not particularly limited, and a conventionally known method can be used. As a specific example, there is a method of immersing a dielectric member having a metal film formed on the surface thereof in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the gold thin film to form a SAM.

  In addition, instead of forming SAM, a “dielectric spacer layer” may be formed. In this case, a silane having two or more different reactive groups in the molecule is a compound composed of an organic substance and silicon. A coupling agent may be used. A silane coupling agent is a molecule having an ethoxy group (or methoxy group) that gives a silanol group [Si-OH] by hydrolysis, and a reactive group such as an amino group, glycidyl group, or carboxyl group at the other end. it can.

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

  Examples of the method for forming a spacer layer made of a dielectric material include a sputtering method, an electron beam evaporation method, a thermal evaporation method, a formation method by a chemical reaction using a material such as polysilazane, or an application with a spin coater.

  In addition, solid phase layers formed on the SAM include glucose, carboxymethylated glucose, vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic acid. Polymers composed of at least one monomer selected from the group consisting of monomers included in diesters, maleic diesters, fumaric diesters, allyl compounds, vinyl ethers and vinyl ketones Hydrophilic polymers such as dextran and dextran derivatives and vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic diesters, maleic diesters Kind More preferably, it contains a hydrophobic polymer composed of a hydrophobic monomer included in each of diesters, allyl compounds, vinyl ethers and vinyl ketones, and dextran such as carboxymethyl dextran [CMD] is a living body. It is particularly suitable from the viewpoints of affinity, suppression of nonspecific adsorption reaction, and high hydrophilicity.

The average film thickness of the solid phase layer is preferably 3 nm or more and 80 nm or less. This film thickness can be measured using an atomic force microscope [AFM] or the like. When the average film thickness of the solid phase layer is within such a range, it is preferable that the signal of the assay is stabilized and increased when the SPFS sensor chip is used in the assay method.
2. Calibration curve used for quantitative measurement A method for creating a calibration curve used when quantifying an analyte having a specific sugar chain using the quantitative measurement apparatus 10 described above will be described in detail.

  First, in the SPR measurement unit 28 of the quantitative measurement device 10, incident light 22 is emitted from the light source 20 to the metal film 14 through the dielectric member 12, and the metal film reflected light 24 is received by the light receiving means 26.

  In this state, the signal (SPR signal) of the metal film reflected light 24 received by the light receiving means 26 is measured while changing the incident angle α1 of the incident light 22 by the incident angle adjusting means.

  Then, an incident angle α2 (an incident angle for ATR) that minimizes the SPR signal is found, and the incident angle adjusting unit is fixed so that the incident angle of the incident light 22 becomes α2.

  Subsequently, an appropriate amount of standard antigen with a predetermined concentration is fed into the flow path 36 provided in the sensor chip 16 and circulated for a predetermined time. Here, as the standard antigen, for example, control L for Mucoswaco AFP-L3 can be used. Moreover, the density | concentration of a standard antigen can be adjusted by diluting with phosphate buffered saline (PBS) etc., for example.

  Simultaneously with feeding the standard antigen, the SPR measurement is started in the SPR measurement unit 28 of the quantitative measurement apparatus 10. That is, incident light 22 from the light source 20 is applied to the metal film 14 via the dielectric member 12, and the metal film reflected light 24 is received by the light receiving means 26.

  Subsequently, for example, a cleaning liquid such as TBS (Tris-Buffered Saline) containing Tween 20 is sent to the flow path 36, and the light amount of the metal film reflected light (hereinafter referred to as “SPR signal”) by SPR measurement after a predetermined time has elapsed. Measure).

  Next, an appropriate amount of a lectin solution containing a lectin that is labeled with a fluorescent dye and binds to a specific analyte as a fluorescently labeled probe is fed to the flow path 36 and circulated for a predetermined time. Here, as the fluorescent dye, for example, Alexa Fluor 647 can be used.

  Subsequently, for example, a cleaning liquid such as TBS containing Tween 20 is supplied to the flow path 36, and cleaning is performed for a predetermined time. Then, the SPFS measurement unit 34 performs SPFS measurement. In other words, incident light 22 from the light source 20 is irradiated onto the metal film 14 through the dielectric member 12 and the fluorescence 30 is received by the light detection means 32, whereby the amount of fluorescence by SPFS measurement (hereinafter referred to as “SPFS”). Called “signal”).

  By measuring the SPR signal and the SPFS signal respectively for a plurality of concentrations of the standard antigen in the same manner as described above, a calibration curve relating to the total analyte concentration of the standard antigen and the SPR signal, and specification of the standard antigen A calibration curve for the analyte concentration and the SPFS signal can be generated.

  In addition, a known method can be used to create a calibration curve related to the SPR signal and the SPFS signal. For example, from the obtained SPR signal or SPFS signal, linear approximation, linear interpolation, quadratic interpolation, or the like can be used. A calibration curve can be created.

  The SPR signal and SPFS signal may be used to create a calibration curve using the obtained values as they are, or a blank signal is measured as described later, and an SPR signal value that is the difference between the SPR signal and the blank signal. A calibration curve can also be created using the SPFS signal value which is the difference between the SPFS signal and the blank signal.

The calibration curve relating to the SPR signal and the SPFS signal obtained in this way is stored in advance in the quantitative calculation means 40, whereby an unknown sample can be quantified as follows.
3. Quantitative measurement method First, an appropriate amount of an unknown sample is fed into the flow path 36 provided in the sensor chip 16 and circulated for a predetermined time. Simultaneously with feeding the unknown sample, the SPR measurement is started in the SPR measurement unit 28 of the quantitative measurement device 10.

  Subsequently, the cleaning liquid is sent to the flow path 36, and the SPR signal after a predetermined time has elapsed is measured. The SPR signal obtained here is sent to the quantitative calculation means 40, and the total analyte concentration contained in the unknown sample is calculated based on the calibration curve related to the SPR signal.

  Next, an appropriate amount of a lectin solution labeled with a fluorescent dye and containing a lectin that binds to a specific analyte is fed to the flow path 36 and reacted for a predetermined time.

  Subsequently, the cleaning liquid is supplied to the flow path 36 and cleaning is performed for a predetermined time. Then, the SPFS measurement unit 34 measures the SPFS signal. The SPFS signal obtained here is sent to the quantitative calculation means 40, and the specific analyte concentration contained in the unknown sample is calculated based on the calibration curve related to the SPFS signal.

  Based on the total analyte concentration and the specific analyte concentration thus obtained, the quantitative calculation means 40 uses the calculation formula shown in Equation 1 to calculate the ratio of the specific analyte to the total analyte amount (hereinafter simply referred to as “ Also referred to as “specific analyte percentage (%)”.

(Configuration of quantitative measurement device)
The quantitative measurement apparatus used in this example has basically the same configuration as the quantitative measurement apparatus 10 described above.

  Note that a laser diode (LD) capable of irradiating light with a wavelength of 635 nm is used as the light source 20, and a neutral density filter (neutral density filter) is used as an optical filter between the light source 20 and the dielectric member 12. ) To adjust the photon amount.

  Further, as the dielectric member 12, a 60-degree prism manufactured by Sigma Kogyo Co., Ltd. is used, and a sensor chip 16 is configured by fixing a plasmon excitation sensor, which will be described later, to the upper portion of the dielectric member 12. .

Further, an objective lens is provided as a condensing lens above the sensor chip 16, and a photomultiplier tube (PMT) is used as the light detection means 32.
(Production of plasmon excitation sensor)
A glass transparent flat substrate (S-LAL 10 manufactured by OHARA INC.) Having a refractive index of 1.72 and a thickness of 1 mm was plasma-cleaned, and a chromium thin film was formed on one surface of this substrate by a sputtering method. Thereafter, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 44 to 52 nm.

  Thus, the board | substrate with which the gold thin film was formed was immersed in the ethanol solution which contains 10 mM of 10-carboxy- 1-decanethiol for 24 hours or more, and the SAM film was formed on the surface of the gold thin film. The substrate was removed from this solution, washed with ethanol and isopropanol, and then dried using an air gun.

A sheet made of polydimethylsiloxane (PDMS) having a groove serving as a flow channel with a height of 0.5 mm and having through holes at both ends of the groove is formed on the groove so that the surface of the SAM film is inside the flow channel. The PDMS sheet (flow path 36) and the plasmon excitation sensor were fixed with screws using pressure from the upper part of the PDMS sheet outside the flow path.
(Preparation of labeled lectin)
A fluorescently labeled lectin was prepared using a fluorescent substance labeling kit. LCA lectin equivalent to 100 μg, 0.1 M sodium bicarbonate and Alexa Fluor 647 reactive dye were mixed, reacted at room temperature for 1 hour, then subjected to gel filtration chromatography and ultrafiltration, and not used for labeling Alexa Fluor 647 reactive dye was removed. Thereafter, the absorbance was measured to quantify the labeled lectin concentration.
(Solid phase of antibody)
The prepared sensor chip is connected to the external flow path, ultrapure water is used for 10 minutes, and then phosphate buffered saline (PBS) is used for 20 minutes with a peristaltic pump at room temperature (25 ° C.) at a flow rate of 500 μL / min. The liquid was circulated to equilibrate the surface.

Subsequently, 5 mL of phosphate buffered saline (PBS) containing 50 mM N-hydroxysuccinimide (NHS) and 100 mM water-soluble carbodiimide (WSC) was fed and circulated for 20 minutes. The primary antibody is solid-phased on the SAM by circulating 2.5 mL of α-fetoprotein (AFP) monoclonal antibody (1D5, 2.5 mg / mL, manufactured by Japan Medical Laboratory) for 30 minutes. Turned into. In addition, the non-specific adsorption | suction prevention process in a flow path was performed by circulating and feeding the phosphate buffered saline (PBS) containing 1 weight% bovine serum albumin (BSA) for 30 minutes.
(Preparation of calibration curve for SPR signal)
FIG. 3 is an enlarged schematic view schematically showing the sensor unit 38 before feeding the standard antigen (analyte 46), and FIG. 4 shows the sensor unit 38 after feeding the standard antigen (analyte 46). It is an expansion schematic diagram showing typically.

  As shown in FIG. 3, the primary antibody (ligand 44) as described above is formed in the sensor unit 38 before the standard antigen is sent.

  As a standard antigen, a control L for Mutus Wako AFP-L3 (L3 = 32%, AFP concentration 49 ng / mL) was diluted with a phosphate buffered saline (PBS) solution, and an AFP antigen concentration 0 ng / mL. 1.0 ng / mL, 2.0 ng / mL, 4.0 ng / mL, and 8.0 ng / mL calibration curve sample AFP antigen solutions were prepared.

  Each calibration curve sample AFP antigen solution is an antigen solution containing 32% of L3. The concentrations of L3 are 0 ng / mL, 0.32 ng / mL, 0.64 ng / mL, 1.28 ng / mL, 2 .56 ng / mL.

  0.5 mL of the calibration curve sample AFP antigen solution at each concentration was added to the flow path and circulated for 20 minutes. Subsequently, TBS containing 0.05% by weight of Tween 20 (TBS-T) was fed and washed for 15 minutes.

  By feeding the standard antigen (analyte 46) in this way, the calibration curve sample AFP antigen binds to the ligand 44 as the analyte 46 as shown in FIG. Reference numeral 46a denotes a protein of a calibration curve sample AFP antigen, and reference numerals 46b and 46c denote sugar chains of the calibration curve sample AFP antigen. In the present example, the calibration curve sample AFP antigen having the sugar chain 46c is an AFP having a sugar chain structure unique to the specific analyte AFP-L3.

  At the same time as feeding the calibration curve sample AFP antigen solution, the quantitative measurement apparatus 10 causes laser light to enter at a predetermined incident angle (56 ° in this embodiment), and the light reflected by the gold thin film is received by the light receiving means 26. The SPR measurement was performed in real time using a photodiode.

  The SPR signal value that is the difference between the SPR signal of each calibration curve sample AFP antigen solution and the blank signal, with the SPR signal when the calibration curve sample AFP antigen solution having an AFP antigen concentration of 0 ng / mL is fed as a blank signal As shown in FIG. Further, SPR signal values after 10 minutes from washing with TBS are as shown in Table 1 below.

表１にまとめた各検量線用サンプルＡＦＰ抗原溶液のＳＰＲシグナル値を用いて、図６に示すような検量線が得られた。なお、検量線の作成には、線形近似を用いた。
（ＳＰＦＳシグナルに関する検量線の作成）
図７は、蛍光標識プローブ４８を送液した後のセンサ部３８を模式的に表す拡大模式図である。

Using the SPR signal values of the calibration curve sample AFP antigen solutions summarized in Table 1, calibration curves as shown in FIG. 6 were obtained. Note that linear approximation was used to create the calibration curve.
(Preparation of calibration curve for SPFS signal)
FIG. 7 is an enlarged schematic view schematically showing the sensor unit 38 after feeding the fluorescently labeled probe 48.

  After the reaction of each calibration curve sample AFP antigen solution, as a fluorescently labeled probe 48, an LCA lectin solution labeled with Alexa Fluor 647 (dissolved in phosphate buffered saline (PBS) to 1 μg / mL) was used. 1 mL was added, and the solution was fed at a flow rate of 200 μL / min for 5 minutes.

  Thereafter, TBS containing 0.05% by weight of Tween 20 (TBS-T) was fed and washed for 5 minutes. Thereafter, SPFS measurement was performed with a quantitative measurement apparatus.

  In this way, by sending the LCA lectin solution as the fluorescently labeled probe 48, as shown in FIG. 7, the LCA lectin specifically binds to the sugar chain 46c, and only AFP-L3 is selectively fluorescently labeled. can do.

  SPFS signal value, which is the difference between the SPFS signal of each calibration curve sample AFP antigen solution and the blank signal, with the SPFS signal at the time of feeding the calibration curve sample AFP antigen solution having an AFP antigen concentration of 0 ng / mL as the blank signal Was as shown in Table 2 below.

表２にまとめた各検量線用サンプルＡＦＰ抗原溶液のＳＰＦＳシグナル値を用いて、図８に示すような検量線が得られた。なお、検量線の作成には、線形近似を用いた。
（テストサンプルの定量測定）
テストサンプルとして、ＡＦＰ抗原濃度５ｎｇ／ｍＬ、Ｌ３含有量２０％のＡＦＰ抗原溶液を調製した。調製は、ミュータスワコー ＡＦＰ−Ｌ３用コントロールＬ（Ｌ３＝２０％、ＡＦＰ濃度２００ｎｇ／ｍＬ）をリン酸緩衝生理食塩水（ＰＢＳ）によって希釈することで行った。

A calibration curve as shown in FIG. 8 was obtained using the SPFS signal values of the sample AFP antigen solutions for the calibration curves summarized in Table 2. Note that linear approximation was used to create the calibration curve.
(Quantitative measurement of test samples)
As a test sample, an AFP antigen solution having an AFP antigen concentration of 5 ng / mL and an L3 content of 20% was prepared. The preparation was performed by diluting the control L for Mutus Wako AFP-L3 (L3 = 20%, AFP concentration 200 ng / mL) with phosphate buffered saline (PBS).

  For this AFP antigen solution, SPR measurement and SPFS measurement were performed in the same manner as described above. As a result, the SPR signal value was 80 and the SPFS signal value was 20106. When quantification was performed using the calibration curve for the SPR signal and the calibration curve for the SPFS signal, the total analyte concentration was 4.61 and the L3 concentration was 1.03. By substituting these values into Equation 1, the ratio of L3, which is a specific analyte, is 22%.

  As described above, the result of the concentration determination was within ± 5%, and the result of the L3 ratio almost coincided with the measured value and the theoretical value was obtained. Thus, by using the quantitative measurement method of the present invention, for example, the amount of sugar chains in the serum of cancer patients can be accurately and rapidly quantified.

  The preferred embodiment of the present invention has been described above, but the present invention is not limited to this. For example, in the above embodiment, the light source of the SPR measurement unit and the light source of the SPFS measurement unit are shared. Although it is covered by one light source, a plurality of light sources may be provided as necessary.

  In addition, although the mode of circulating and feeding various solutions has been described as an example, it is not always necessary to circulate. Various modifications can be made without departing from the object of the present invention, such as an aspect in which the liquid is retained for a predetermined time after the liquid is added.

  INDUSTRIAL APPLICABILITY The present invention enables accurate and rapid quantitative measurement of a specific analyte in a field where high-precision measurement is required, such as clinical tests such as AFP sugar chain measurement and CEA sugar chain measurement.

DESCRIPTION OF SYMBOLS 10 Quantitative measurement apparatus 12 Dielectric member 12a Upper surface 12b Side surface 12c Side surface 14 Metal film 14a Upper surface 16 Sensor chip 18 Sensor chip loading part 20 Light source 22 Incident light 24 Metal film reflected light 26 Light receiving means 28 SPR measurement part 30 Fluorescence 32 Light detection means 34 SPFS measurement unit 36 Fine channel 38 Sensor unit 40 Quantitative calculation means 44 Ligand 46 Analyte 46a Protein 46b Sugar chain 46c Sugar chain 48 Fluorescently labeled probe

Claims (6)

A quantitative measurement method for quantifying a specific analyte,
A sensor chip having a dielectric member, a metal film formed on the dielectric member, and a ligand that captures an analyte including the specific analyte formed on the metal film is provided on the sensor chip. Providing a specimen containing light and contacting the ligand;
The metal film of the sensor chip is irradiated with incident light through the dielectric member, and the metal film reflected light reflected by the metal film is received by a light receiving unit, thereby receiving the metal. Measuring the amount of film reflected light; and
After measuring the amount of light reflected from the metal film, a step of reacting a fluorescently labeled probe for fluorescently labeling the specific analyte with the specific analyte;
Fluorescence generated by exciting the fluorescently labeled probe bound to the specific analyte with surface plasmon light generated by irradiating incident light through the dielectric member to the metal film of the sensor chip Measuring the amount of the fluorescence by receiving the light by the light detection means,
Calculating the total analyte concentration in the specimen based on the amount of light reflected by the metal film;
Calculating a specific analyte concentration in the specimen based on the amount of fluorescence;
Calculating a ratio of a specific analyte amount to a total analyte amount from the total analyte concentration and the specific analyte concentration;
A quantitative measurement method comprising:   The quantitative measurement method according to claim 1, wherein a channel is formed on the metal film, and the ligand is formed in a part of the channel.   The quantitative measurement method according to claim 1, wherein the ligand is a protein or a nucleic acid.   The quantitative measurement method according to claim 1, wherein the specific analyte has a sugar chain.   The quantitative measurement method according to claim 4, wherein the fluorescently labeled probe is a lectin that is labeled with a fluorescent dye and binds to the specific analyte. The step of calculating the total analyte concentration in the specimen is to measure a calibration curve relating to the total analyte concentration and the amount of reflected light of the metal film, and the amount of reflected light of the metal film, from a plurality of standard antigens. While performing by calculating based on the amount of reflected light of the metal film measured in the process,
The step of calculating a specific analyte concentration in the specimen is measured by a calibration curve relating to a specific analyte concentration and a fluorescence light amount prepared in advance from a plurality of standard antigens, and a step of measuring the fluorescence light amount. 6. The quantitative measurement method according to claim 1, wherein the quantitative measurement method is performed based on a calculation based on the amount of fluorescence.

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