JP2012233860A - Assay method of object to be analyzed in biological specimen and poct apparatus used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel assay method of an object to be analyzed in a biological specimen applicable as a measuring method for a POCT apparatus.SOLUTION: The assay method of an object to be analyzed in a biological specimen includes the steps of: bringing a biological specimen, a specific binding partner A that specifically binds to an object to be analyzed in the biological specimen and a fluorescent substance into contact with a sensor chip, wherein the fluorescent substance is bound to the specific binding partner A or is capable of specifically binding to the specific binding partner A, the sensor chip includes a base plate, a metal film formed on the surface of the base plate and a specific binding partner B fixed on the metal film, the specific binding partner B is specifically bound to a portion of the object to be analyzed different from a bound portion of the specific binding partner A; irradiating the sensor chip with focused incident light from the opposite surface of the base plate formed with the metal film through a prism so as to generate a surface plasmon resonance on the metal film; and receiving fluorescent from the fluorescent substance excited by the surface plasmon resonance.

Description

  The present invention relates to an assay method for an analyte in a biological sample and a POCT apparatus used therefor.

  Various tests using biological samples such as blood or urine are performed for the detection and prevention of diseases. This test is usually performed in a concentrated manner in a specialized organization such as a clinical laboratory center because of the advantages that the measurement is accurate and various measurements can be performed at once. However, when performing examinations in such a concentrated manner at specialized institutions, work such as collection and delivery of samples collected at hospitals, etc. is required, so the demerit that it takes time to obtain test results, There is a demerit that the amount of the sample required for inspection increases. For this reason, in recent years, effective utilization of POC (Point of Care) has been demanded. POC refers to a clinical test performed by a patient in a clinical setting. If various tests can be performed by a patient using a POCT (Point of Care Testing) device, a quick diagnosis by a doctor and / or The treatment is possible, and the efficiency and speed of medical care and treatment can be expected.

  In POC, since simple and quick measurement is required, it is desired to use a sample collected from a patient as it is without separation / purification, for example. For this reason, in a measurement using a POCT instrument, there is a demand for a method that is easy to operate and can be detected at high speed and high sensitivity even for a sample that is not subjected to separation / purification.

  On the other hand, biosensing using surface plasmon resonance (SPR) has attracted attention. SPR is an interaction that occurs between a surface plasmon (elastic wave) generated on the surface of a metal film and an evanescent wave (light wave) generated by a totally reflected electromagnetic wave. By using this SPR, a minute change in the refractive index in the vicinity of the metal film can be detected, so that it is possible to quantify a trace component in a test sample by an immunoassay utilizing an antigen-antibody reaction. As another method using SPR, for example, there is a surface plasmon excitation fluorescence method (SPFS). SPFS is a method for exciting a fluorescent substance with high efficiency using an enhanced electric field generated on the surface of a metal film when SPR occurs. Thus, since SPFS is excited and excited to detect fluorescence, it can detect a trace amount and / or a very low concentration of an analyte, for example, contained in a sample such as blood or urine. Attempts have been made to detect such tumor markers and nucleic acids (Patent Document 1, etc.).

JP 2010-271124 A

  The inventors of the present invention pay attention to the fact that if a biomarker such as a tumor marker can be measured immediately in a clinical setting, a rapid diagnosis and / or treatment by a doctor will be possible, and the efficiency and speed of medical treatment / treatment can be expected. We have attempted to develop a new measurement method that can be used as a POCT instrument using the SPFS measurement principle. For the measurement of SPFS, it is important that light is incident on the metal film at an angle satisfying the resonance condition between the surface plasmon generated on the metal film surface and the evanescent wave. However, when a sample collected from a patient is used as it is without separation / purification, the refractive index of the sample is different for each test sample as a result of the concentration of the protein or salt contained in the test sample being different. This change in refractive index affects the resonance conditions and thus greatly affects the measurement signal. For this reason, in order to perform detection with high sensitivity, it is necessary to confirm and adjust the resonance conditions for each test sample, which makes the operation extremely complicated and requires time for measurement. On the other hand, since a POCT device is used in a clinical setting, rapid measurement, downsizing, and easy operation are required.

  Therefore, the present invention provides a new assay method for an analyte in a biological sample that can be used as a measurement method in a POCT instrument.

  The present invention relates to a method for assaying an analyte in a biological sample, wherein a biological sample, a specific binding partner A that specifically binds to the analyte in the biological sample, and a fluorescent substance are contacted with a sensor chip. Here, the fluorescent substance is bound to the specific binding partner A or can be specifically bound to the specific binding partner A, and the sensor chip is specific to the substrate and the metal film. A binding partner B, wherein the metal film is formed on the substrate surface, and the specific binding partner B specifically binds to a site of the analyte different from a binding site of the specific binding partner A. A binding partner, which is immobilized on the metal film; the gold on the sensor chip from the surface of the substrate opposite to the surface on which the metal film is formed via a prism. The surface plasmon resonance in the film is irradiated by converging the incident light to generate; and relates to an assay method comprising receiving fluorescence of the fluorescent substance excited by the surface plasmon resonance.

  ADVANTAGE OF THE INVENTION According to this invention, the new assay method of the analysis target substance in a biological sample which can be utilized as a measuring method in a POCT apparatus can be provided.

FIG. 1 is a configuration diagram illustrating an example of an SPFS apparatus according to an embodiment of the present invention. FIG. 2 is a conceptual diagram illustrating an example of a measurement method according to an embodiment of the present invention. FIG. 3 is a configuration diagram illustrating another example of the SPFS apparatus according to the embodiment of the present invention. FIG. 4 is a graph showing an example of the results of Example 1 and Comparative Example 1. FIG. 5 is a graph showing an example of the result of Comparative Example 2. FIG. 6 is a graph showing an example of the result of Example 2.

  In the present invention, a biological sample, a specific binding partner A that specifically binds to an analyte in the biological sample, and a fluorescent substance are brought into contact with a sensor chip in which the specific binding partner B is immobilized on a metal film. After that, by condensing and irradiating light with different incident angles on the sensor chip, the incident angle is adjusted even when the concentrations of proteins and salts contained in each test sample are different. This is based on the knowledge that stable measurement is possible even without this, and that operability is excellent and rapid measurement and downsizing of the apparatus can be possible. In addition, the present invention is based on the knowledge that the above method can be used as the measurement principle of the POCT apparatus because it is excellent in operability and can enable rapid measurement and downsizing of the apparatus.

  According to the assay method of the present invention, since light of various incident angles is condensed and irradiated to the sensor chip so that SPR occurs in the metal film, an operation of adjusting the incident angle for each test sample is performed. It becomes unnecessary. For this reason, according to the assay method of the present invention, even if the refractive index differs for each test sample due to the difference in protein or salt concentration contained in the test sample, it is stable with simple operability. There is an effect that measurement with high sensitivity becomes possible. In addition, according to the assay method of the present invention, it is not necessary to adjust the incident angle, so that quick measurement is possible, and a goniometer or the like for adjusting the incident angle is not required, thereby reducing the size and weight of the apparatus. There is an effect that it becomes possible.

  In this specification, “condensing incident light” includes collecting and irradiating light beams emitted from a light source in a spot shape. By condensing and irradiating incident light in this way, it is possible to irradiate the sensor chip with light having a plurality of incident angles as incident light, which eliminates the need to adjust the incident angle for each test sample. In addition, stable measurement is possible. In this specification, “to collect incident light so that surface plasmon resonance (SPR) occurs” means to collect light so as to include at least an incident angle that satisfies the condition for generating SPR. It means that light is incident so as to have an incident angle within a predetermined range including the incident angle to be satisfied. In this specification, the “incident angle” is an angle between the optical axis and the normal line of the main surface of the sensor chip, for example, an angle indicated by θ in FIG.

  In the present specification, the “biological sample” includes a biological component collected from a subject and is not particularly limited. For example, blood, serum, plasma, urine, nasal fluid and saliva Etc. The biological sample is preferably not separated and / or purified from the viewpoint of easy operability and quick measurement, and more preferably as it is collected from the subject. As used herein, “as-collected state” refers to a state in which separation and / or purification treatment has not been performed after collection from a specimen, including, for example, diluted after being collected from a specimen. You may go out. Examples of the subject include humans and / or mammals other than humans.

  In the present specification, the “analytical object” is contained in a biological sample and is not particularly limited. For example, a nucleic acid (for example, DNA, RNA, polynucleotide, etc.), a protein (poly Peptides, oligopeptides, etc.), amino acids, carbohydrates, lipids, and the like, and specifically include tumor markers, inflammation markers, signal transduction substances, and the like. Although it does not specifically limit as a tumor marker, For example, (alpha) -fetoprotein (AFP), carcinoembryonic antigen (CEA), a prostate specific antigen (PSA) etc. are mentioned. The inflammation marker is not particularly limited, and examples thereof include C-reactive protein (CRP).

  In the present specification, the “fluorescent substance” is a fluorescent label that binds to the specific binding partner A or can bind to the specific binding partner A, and emits fluorescence when excited by surface plasmons. I mean. In the present specification, the fluorescent substance may be the specific binding partner A itself, that is, the specific binding partner A specifically binds to the analyte in the biological sample and emits fluorescence. It may be a substance.

  As used herein, “specific binding partner A” refers to a binding partner that specifically binds to an analyte in a biological sample. Specific binding partner A is not particularly limited, and examples thereof include nucleic acids (for example, DNA, RNA, and polynucleotide), proteins (polypeptide, oligopeptide, and the like), amino acids, carbohydrates, and lipids. Etc. Although it does not specifically limit as protein, For example, the antibody etc. which recognize an analysis object specifically are mentioned. Although it does not specifically limit as an antibody, For example, a polyclonal antibody, a monoclonal antibody, an antibody fragment, etc. are included. Specific binding partner A is preferably one that can be used for antigen-antibody reaction, and more preferably one that is used for sandwich immunoassay.

  As used herein, “specific binding partner B” refers to a partner that specifically binds to a site of an analyte different from the binding site of specific binding partner A. Examples of the specific binding partner B include those similar to the specific binding partner A.

  In this specification, the “sensor chip” refers to a chip including at least a substrate, a metal film formed on the substrate surface, and a specific binding partner B immobilized on the metal film. The sensor chip can trap the analyte by the specific binding partner B immobilized on the metal film, for example, using an antigen-antibody reaction. In the present specification, “specific binding partner B immobilized on a metal film” means that the specific binding partner B is immobilized via a layer other than the metal film formed on the surface of the metal film, And the specific binding partner B may be directly immobilized on the metal film surface. The layer other than the metal film is not particularly limited, and examples thereof include a self-assembled monomolecular film described later and a spacer layer such as a polymer film. The substrate is preferably a planar support capable of forming a metal film on the surface. The substrate is preferably transparent. The material of the substrate is not particularly limited, and examples thereof include glass, quartz glass, and plastic. Although it does not specifically limit as a plastic, For example, a polycarbonate, a cycloolefin polymer, a polyethylene terephthalate etc. are mentioned. The substrate preferably has a high refractive index. For example, the refractive index of the substrate is preferably 1.5 or more, and more preferably 1.5 to 2.2. The metal film is preferably formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum. The metal film may be formed of one type of metal film, or may be a laminate of a plurality of metal films of different materials. The metal film may be formed on the substrate through an adhesive layer formed of another metal. Examples of the material for the adhesive layer include chromium and titanium. As the sensor chip, a known sensor chip used for SPR or SPFS may be used. The sensor chip may be a prism integrated sensor chip integrated with a prism described later.

  The sensor chip may further have a self-assembled monomolecular film and / or polymer film formed on the surface of the metal film. In this case, the specific binding partner B is preferably immobilized on a self-assembled monolayer or polymer membrane. Although it does not specifically limit as a molecule | numerator which forms a self-assembled monolayer, For example, alkanethiol etc. are mentioned. Examples of the self-assembled monolayer include alkanethiol monolayers having reactive functional groups. Examples of the reactive functional group include a carboxyl group, an amino group, an aldehyde group, an imide group, an acid chloride, an epoxy group, and an isocyanate group.

[Assay Method]
One aspect of the present invention is that a biological sample, a specific binding partner A that specifically binds to an analyte in the biological sample, and a fluorescent substance are brought into contact with the sensor chip. Condensing and irradiating incident light so that SPR is generated in the metal film from a surface opposite to the surface on which the metal film is formed, and the fluorescence excited by the SPR The present invention relates to a method for assaying an analyte in a biological sample, which comprises receiving fluorescence of a substance. Here, the fluorescent substance is bound to the specific binding partner A or can specifically bind to the specific binding partner A. The sensor chip includes a substrate, a metal film, and a specific binding partner B. The metal film is formed on the surface of the substrate, and the specific binding partner B is a binding site of the specific binding partner A. A specific binding partner that specifically binds to a different site of the analyte, and is immobilized on the metal film.

  The order in which the biological chip, the specific binding partner A that specifically binds to the analyte in the biological sample and the fluorescent substance, and the sensor chip are brought into contact with each other is not particularly limited. The binding partner A and the biological sample may be contacted and then contacted with the sensor chip. First, the biological sample is contacted with the sensor chip, and then the specific binding partner A to which the fluorescent substance is bound is used as the sensor chip. You may make it contact.

  The biological sample is preferably used without separating and / or purifying the biological sample collected from the subject. That is, it is preferable that a biological sample collected from a subject is brought into contact with the sensor chip without performing separation and / or purification treatment.

  The irradiation of incident light preferably includes focusing light rays having different incident angles on the sensor chip. The incident light preferably includes light having an angle between the central axis of the light beam and the outside of the light beam (θ ′ in FIG. 1) in the range of 10 degrees or less, so that sufficient fluorescence intensity can be obtained and the apparatus can be downsized. From the point, it is more preferable to include light in a range of 5 degrees or less. In addition, the incident light preferably includes light having an angle (θ ′) between the light beam central axis and the light beam outer side of 3 degrees or more, more preferably light of 4 degrees or more from the viewpoint of performing more stable measurement. including. The incident light preferably includes a light beam having an incident angle (θ in FIG. 1) in the range of 50 to 70 degrees. From the viewpoint of obtaining sufficient fluorescence intensity, the light beam having an incident angle in the range of 55 to 65 degrees. It is preferable to contain.

  The assay method of the present invention preferably includes quantifying the analyte based on the received fluorescence intensity.

  The assay method of the present invention may include assaying an analyte using biological samples collected from different subjects. When an assay is performed using a plurality of biological samples collected from different subjects, the refractive index may vary from sample to sample. However, the assay method of the present invention collects light of various incident angles. Therefore, stable measurement can be performed with a simple operation even in such a case.

[POCT equipment]
In another aspect, the present invention is an apparatus for performing the assay method of the present invention, a light source for generating SPR, a light collecting unit for condensing light from the light source on a sensor chip, The present invention relates to a POCT apparatus including a fluorescence light receiving unit that receives fluorescence excited by SPR generated by irradiation of light condensed by a light collecting unit.

  The POCT apparatus of the present invention preferably further includes a reflected light receiving unit that receives reflected light from the sensor chip.

  Embodiments of the present invention will be specifically described below with reference to the drawings. However, the following description is merely an example, and it goes without saying that the present invention is not limited to this.

[First Embodiment]
FIG. 1 shows an example of an SPFS apparatus in the first embodiment. The SPFS apparatus according to the first embodiment includes a light source 1, a parallel light creating unit 7, a polarizing optical element 8, a condensing unit (condensing lens) 2, a prism 4, and a fluorescence light receiving unit 3.

  The light source 1 emits light for generating SPR in a metal film formed on the sensor chip to excite a fluorescent substance. The wavelength of the light source 1 can be appropriately determined according to the metal film formed on the sensor chip. When the metal film is a gold film, it is, for example, 600 nm or more, preferably 600 to 700 nm. The light source 1 is not particularly limited, and for example, a point light source LED and an LD laser can be used.

  The parallel light creation unit 7 converts the light from the light source 1 into parallel light. In the configuration shown in FIG. 1, the parallel light creating unit 7 is configured by the diaphragm 71 and the lens 72, but is not limited thereto.

  The polarizing optical element 8 is for transmitting p-polarized light that efficiently generates SPR out of the light from the light source 1. As the polarizing optical element 8, for example, an optical element capable of transmitting p-polarized light can be used.

  The condensing unit 2 is for condensing the parallel light beam formed by the parallel light generating unit 7, and is preferably for condensing at one point on the central axis of the light beam. As the condensing part 2, a well-known condensing optical system can be used. The condensing unit 2 is preferably arranged so that light with an angle between the central axis of the light beam and the outside of the light beam (θ ′ in FIG. 1) in a range of 3 degrees to 10 degrees can be emitted. From the viewpoint of more efficient excitation, it is more preferable that the light source is disposed so as to be able to irradiate light in a range of 5 degrees or less.

  The prism 4 is for efficiently irradiating the sensor chip with the light collected by the light collecting unit 2. A sensor chip 5 can be disposed on the prism 4. The prism 4 is not particularly limited, and examples thereof include a triangular prism and a semicircular prism. The material of the prism 4 is not particularly limited, and examples thereof include the same material as that of the sensor chip described above.

  The fluorescence light receiver 3 receives fluorescence excited by surface plasmons. The fluorescence light receiving unit 3 is arranged on the upper part of the sensor chip 5 arranged on the prism 4 so that the excited fluorescence can be received. The fluorescent light receiving unit 3 is not particularly limited, and examples thereof include those capable of detecting weak light such as a CCD camera and a photomultiplier tube. The fluorescence light receiving unit 3 may further include an optical system for receiving fluorescence from the sensor chip 5. For example, as shown in FIG. 1, the fluorescence light receiving unit 3 may include an objective lens 33, a fluorescence filter 32, and a CCD camera 31.

  The fluorescence light receiving unit 3 may be connected to a computer 9 as shown in FIG. Thereby, the output of the fluorescence detected by the light receiving unit 3 and the determination of the analysis object based on the obtained fluorescence intensity can be performed.

  An assay using the SPFS apparatus shown in FIG. 1 will be described with reference to FIGS. FIG. 2 is a schematic diagram showing one embodiment of the assay method of the present invention. In FIG. 2, the same members as those in FIG.

  First, as shown in FIG. 2A, the sensor chip 5 is prepared, and the sensor chip 5 is arranged on the prism 4. The sensor chip 5 is not particularly limited as long as a metal film (not shown) is formed on the surface of a substrate (not shown) and a specific binding partner B11 is bound on the metal film. , Known sensor chips that can be used for SPR or SPFS are available. The thickness of a board | substrate is not specifically limited, For example, it is 0.01-10 mm, Preferably it is 0.5-5 mm. The thickness of the metal film can be appropriately determined according to the material contained in the metal film, and is not particularly limited. For example, the thickness is 70 nm or less, preferably 20 to 70 nm, and more preferably 30 to 60 nm. In the sensor chip, the specific binding partner B11 is preferably immobilized on the metal film via a spacer layer (not shown) from the viewpoint of avoiding the phenomenon that the fluorescence does not occur (metal quenching). . The thickness of the spacer layer can be appropriately determined according to the thickness of the metal film, etc., but is preferably, for example, 20 nm or more, more preferably 25 nm or more, and further preferably 30 nm or more from the viewpoint of avoiding the above-described metal quenching. is there. Specific binding partner B11 can be suitably determined according to the analyte to be measured.

  A flow path 6 may be formed in the sensor chip 5. The sensor chip 5 is preferably provided with a flow path 6 from the viewpoint of efficient delivery of a small amount of chemical solution and reaction promotion, and the sensor chip 5 is not provided with the flow path 6 from the viewpoint of miniaturization of the apparatus. Is preferred.

  Next, the specific binding partner A, the fluorescent substance, and the biological sample are mixed. As a result, the specific binding partner A is bound to the analyte in the biological sample. From the viewpoint of shortening the measurement time and easy operability, it is preferable to bind the fluorescent substance to the specific binding partner A in advance. The mixing temperature is not particularly limited, and is, for example, 4 to 50 ° C., preferably 10 to 40 ° C. The mixing time is not particularly limited, and is, for example, 0.5 to 30 minutes, preferably 1 to 20 minutes.

  As shown in FIG. 2 (b), a mixed solution containing the analyte 12 and the specific binding partner A 13 to which the fluorescent substance 14 is bound is introduced into the sensor chip 5, and specific binding is performed on the sensor chip 5. The fluorescently labeled analyte is brought into contact via partner A13. As a result, as shown in FIG. 2 (c), the analyte 12 is bound to the specific binding partner B 11 immobilized on the sensor chip 5. The contact temperature is not particularly limited, and is, for example, 4 to 50 ° C., preferably 10 to 40 ° C. The contact time is not particularly limited, and is, for example, 0.5 to 60 minutes, preferably 5 to 30 minutes.

  Next, as shown in FIG. 2 (d), light is emitted from the light source 1 to the sensor chip 5 via the prism 4. The light from the light source 1 passes through the parallel light creating unit 7 and the polarizing optical element 8 to become p-polarized light of the parallel light, and enters the sensor chip 5 at the condensing unit 2 at different incident angles including an incident angle that is an SPR condition. The light is condensed so as to include a component that does. The condensed light is irradiated to the sensor chip 5 via the prism 4 so that surface plasmon resonance occurs in the metal film of the sensor chip 5, and surface plasmon resonance occurs. By this surface plasmon resonance, the fluorescent substance 14 immobilized on the metal film is excited through the specific binding partner B 11, the analyte 12 and the specific binding partner A 13 to emit fluorescence.

  Then, the excited fluorescence is measured in the fluorescence light receiving unit 3. As described above, since the sensor chip 5 is irradiated with light having a plurality of incident angles including the incident angle that is the SPR condition, the liquid mixture introduced into the sensor chip 5 due to a change in protein or salt concentration. Even when the refractive index fluctuates, fluorescence can be measured stably.

  Based on the fluorescence intensity received by the fluorescence light receiver 3, for example, an analysis object in a biological sample can be quantitatively analyzed.

[Second Embodiment]
FIG. 3 shows an example of the SPR-SPFS apparatus in the second embodiment. In FIG. 3, the same members as those in FIG. The configuration of the SPR-SPFS apparatus in the second embodiment is the same as that of the SPFS apparatus of the first embodiment except that the reflected light receiving unit 10 is provided.

  The reflected light receiving unit 10 receives light reflected at the interface between the sensor chip 5 and the prism 4, and is disposed below the sensor chip 5, that is, on the prism 4 side. The reflected light receiving unit 10 is not particularly limited, and examples thereof include a CCD camera and a line sensor. The reflected light receiving unit 10 may further include a condensing optical system 102 for condensing the light reflected at the interface between the sensor chip 5 and the prism 4. The reflected light receiving unit 10 may be constituted by, for example, a condenser lens 102 and a CCD camera 101 as shown in FIG. The reflected light receiving unit 10 may be connected to the PC 9 as shown in FIG. Thereby, the fluorescence detected by the reflected light receiving unit 10 can be output.

  Since the SPR-SPFS apparatus in the second embodiment includes the reflected light receiving unit 10 in addition to the fluorescence receiving unit 3, the reflected light can be measured together with the fluorescence measurement.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the present invention is not limited to the following examples.

Example 1
The analyte in the test sample was assayed using the method of the present invention. As test samples, physiological saline containing bovine serum albumin (BSA) at different concentrations (3%, 7% and 10%) and the analyte was prepared. Α-fetoprotein (AFP), which is a hepatocellular carcinoma marker, is used as an analysis target, an anti-AFP antibody (Mikuri Immuno Laboratory: Clone 6D2) is used as a specific binding partner A, and Alexa647 (Invitrogen) is used as a fluorescent substance. The anti-AFP antibody (Mikuri Immuno Laboratory: Clone 1D5) was used as the specific binding partner B (primary antibody that binds to AFP).

[Fabrication of sensor chip]
Chromium is vapor-deposited on one side of a glass substrate (optical glass: S-LAL10, 0.5 mm) to form a chromium film (thickness: 1 nm), and gold is vapor-deposited thereon to form a gold film (thickness: 49 nm). did. Subsequently, a self-assembled monomolecular film (COOH-SAM, thickness: 2 nm) having a carboxylic acid at the terminal was formed on the gold film using 11-Mercaptoundecanonc acid (HS (CH ₂ ) ₁₀ COOH). The active ester was introduced into the substrate on which COOH-SAM was formed using the carbodiimide method, and the specific binding partner B (10 μg / mL) was immobilized.

[Configuration of SPFS device]
In this example, the SPFS apparatus shown in FIG. 1 was used. As shown in FIG. 1, the SPFS device includes a light source 1, a parallel light creating unit 7, a polarizing optical element 8, a condensing unit (condensing lens) 2, a prism 4, a light receiving unit 3, and a computer 9. A sensor chip 5 including a flow path 6 can be disposed on the prism 4. The parallel light generator 7 is formed by a diaphragm 71 and a lens 72. The light receiving unit 3 includes an objective lens (× 20) 33, a fluorescent filter 32, and a CCD camera 31, and the CCD camera 31 is connected to the computer 9. The light from the light source 1 is converted into parallel light by the parallel light creating unit 7, and then collected by the light collecting unit 2 and then irradiated to the interface between the prism 4 and the sensor chip 5. In the light source 1, the parallel light creating unit 7, the polarizing optical element 8, and the light collecting unit 2, the incident light beam irradiated from the light source 1 to the interface between the prism 4 and the sensor chip 5 is 55 to 65 degrees with respect to the sensor chip 5. It is arranged to become. As the light source 1, a point light source LED (wavelength 650 nm) was used. As the prism 4, a triangular prism (optical glass: S-LAL10, refractive index = 1.72) was used.

[Analysis of analyte]
A specific binding partner A (fluorescent labeled anti-AFP antibody) labeled with a fluorescent substance was added to the test sample to prepare a sample (final concentration of AFP in the sample: 5 ng / mL). As the fluorescently labeled anti-AFP antibody, a monoclonal antibody that recognizes an epitope different from the antibody immobilized on the sensor chip was used. After the sensor chip 5 including the flow path 6 is arranged on the prism 4 of the SPFS apparatus, each prepared specimen is introduced into the flow path 6 and light is irradiated from the light source 1 to the interface between the prism 4 and the sensor chip 5 to fluoresce. Was measured. The result is shown in FIG. 3 as a relative value where the result of 7% BSA is 1.

(Comparative Example 1)
Fluorescence measurement was performed in the same manner as in Example 1 except that the incident angle of light applied to the sensor chip was set to 60 degrees (one point). The results are shown in FIG. 4 together with the results of Example 1.

  FIG. 4 is a graph showing the rate of increase in fluorescence intensity. FIG. 4 (a) is a graph of Example 1, and FIG. 4 (b) is a graph of Comparative Example 1. As shown in FIG. 4B, in Comparative Example 1 in which the incident angle was fixed at one point and a single incident angle was incident, the rate of increase in the fluorescence signal greatly changed depending on the concentration of BSA. On the other hand, as shown in FIG. 4A, in Example 1 in which light beams having different incident angles are condensed and irradiated, the fluorescence signal accompanying the change in the concentration of BSA is compared with Comparative Example 1. There was almost no fluctuation in the ascent rate. Therefore, by collecting and irradiating light beams having different incident angles, it can be confirmed that stable measurement can be performed even when the concentration of contained protein or salt varies depending on the test sample. It was.

  It has been confirmed that when the single incident angle is incident, the rate of increase in the fluorescence signal greatly varies depending on the concentration of the protein contained in the sample even when the coexisting protein is other than BSA. The results are shown in Comparative Example 2.

(Comparative Example 2)
Human serum albumin (HSA) (Sigma, A1653) was used instead of BSA, CRP as the analyte, CRP monoclonal antibody as the specific binding partner A (Immunoprobe, clone No. 8, L / N080317), Except for using CRP monoclonal antibody (Clone No.7, L / N030700, manufactured by Immunoprobe) as specific binding partner B and setting the incident angle of light irradiated to the sensor chip to 59 degrees (one point) Fluorescence measurement was performed in the same manner as in Example 1. The result is shown in FIG. 5 as a relative value with the result of 7% HSA being 1. As shown in FIG. 5, the increase rate of the fluorescence signal greatly changed depending on the concentration of HSA.

(Example 2)
Measurements were performed using the same conditions and apparatus as in Example 1 except that commercially available fetal bovine serum (FBS) containing various concentrations of AFP was used as the test sample. An example of the result is shown in FIG. As shown in FIG. 6, AFP can be quantified at a clinical cut-off value (10 ng / mL) or less even in serum in which a high concentration of protein coexists by condensing and irradiating incident light. It was shown that there is.

  From the above results, according to the assay method of the present invention, even when the concentration of protein or salt contained in each test sample is different, it is possible to perform a stable measurement with a simple operation. It was suggested that it is useful as a measurement method in a POCT instrument.

  The present invention is useful in various fields, such as the medical field and the field of clinical examination.

Claims (10)

An assay method for an analyte in a biological sample, comprising:
Contacting a biological sample, a specific binding partner A that specifically binds to an analyte in the biological sample, and a fluorescent substance with a sensor chip, wherein the fluorescent substance binds to the specific binding partner A Or is capable of specifically binding to the specific binding partner A, the sensor chip includes a substrate, a metal film, and a specific binding partner B, and the metal film is formed on the substrate surface, The specific binding partner B is a specific binding partner that specifically binds to a site of the analyte different from the binding site of the specific binding partner A, and is immobilized on the metal film,
Irradiating the sensor chip by collecting incident light so that surface plasmon resonance occurs in the metal film via a prism from a surface opposite to the surface on which the metal film is formed on the substrate; as well as,
Receiving the fluorescence of the fluorescent material excited by the surface plasmon resonance. The assay method according to claim 1, wherein the irradiation of the incident light includes focusing light beams having different incident angles on the sensor chip. The assay method according to claim 1 or 2, wherein the irradiation of the incident light includes condensing a light beam having an incident angle in a range of 50 to 70 degrees on the sensor chip. The assay method according to any one of claims 1 to 3, comprising quantifying the analyte based on the received fluorescence intensity. The assay method according to any one of claims 1 to 4, wherein the biological sample includes at least one selected from the group consisting of blood, serum, plasma, urine, nasal fluid and saliva. The assay method according to any one of claims 1 to 5, comprising bringing a biological sample collected from a subject into contact with the sensor chip without performing separation and / or purification treatment. The assay method according to any one of claims 1 to 6, comprising assaying an analyte on biological samples collected from different subjects. The assay method according to claim 1, wherein the metal film is formed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum. An apparatus for performing the assay method according to any one of claims 1 to 8, comprising:
A light source for generating surface plasmon resonance;
A condensing unit for condensing light from the light source on a sensor chip;
A POCT apparatus comprising: a fluorescence light receiving unit that receives fluorescence excited by surface plasmon resonance generated by irradiation of light collected by the light collecting unit. The POCT apparatus according to claim 9, further comprising a reflected light receiving unit that receives reflected light from the sensor chip.

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