JP5831177B2 - Measuring method and measuring device - Google Patents

Description

  The present invention relates to a measurement method and a measurement apparatus that perform measurement by surface plasmon excitation fluorescence spectroscopy.

  When excitation light traveling inside the dielectric medium is incident on the interface between the metal film and the dielectric medium while satisfying the total reflection condition, evanescent waves leak from the interface, and plasmons and evanescent waves on the surface of the metal film leak out. Interfere with. When the incident angle of the excitation light to the interface is set to the resonance angle and the plasmon and the evanescent wave resonate, the electric field of the evanescent wave is remarkably enhanced. This enhanced electric field is used in measurements by surface plasmon excitation fluorescence spectroscopy (SPFS).

  In the measurement by SPFS, an antigen capturing film is formed on the surface of the metal film, the antigen is captured by the antigen capturing film, and a fluorescent label is added to the captured antigen. The enhanced electric field excites the fluorescent label, and surface plasmon excited fluorescence is emitted from the fluorescent label. The amount of surface plasmon excitation fluorescence is measured, and the presence or absence of the antigen, the concentration of the antigen, etc. are determined.

  Patent Document 1 relates to detection of a resonance angle in measurement by surface plasmon excitation fluorescence. In the detection of the resonance angle, the incident angle of the excitation light to the interface is scanned.

JP 2004-354092 A

  In the measurement by SPFS, the type of the antigen capturing film is selected according to the type of antigen to be measured. For example, the type and density of the antibody immobilized on the antigen capturing film are selected according to the type of antigen to be measured.

  On the other hand, when the type of the antigen capture membrane is changed, the resonance angle is also changed when other measurement conditions are the same.

  For these reasons, when the type of antigen to be measured changes, the resonance angle also changes. When the resonance angle changes, in the detection of the resonance angle, the scanning range of the incident angle of the excitation light to the interface is widened, making it difficult to detect the resonance angle. For example, problems such as an increase in time required for measurement and a complicated apparatus required for measurement occur.

  The present invention is made to solve these problems. An object of the present invention is to provide a measurement method and a measurement apparatus in which a difference in resonance angle due to a difference in the type of an antigen capture film is reduced and the resonance angle is easily detected.

  The first to fourth aspects of the present invention are directed to a measurement method that performs measurement by surface plasmon excitation fluorescence spectroscopy.

  In the first aspect of the present invention, the difference in the resonance angle due to the difference in the type of the antigen capture membrane in two or more types of antigen capture membranes and two or more types of measurement liquids in which the antibody that binds to the antigen to be measured is immobilized. The combination of the type of the antigen capture membrane and the type of the measurement solution is determined so as to eliminate the problem.

  A specific type of antigen capture membrane is selected from two or more types of antigen capture membranes. A specific type of measurement liquid combined with a specific type of antigen-capturing membrane in the determined combination is selected. A sensor chip having a specific type of antigen capture membrane is prepared.

  The sensor chip includes a dielectric medium, a conductor film, a specific type of antigen capturing film, and a flow path formation. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The incident surface, the reflecting surface, and the emitting surface are arranged so that the excitation light enters the incident surface, is reflected by the reflecting surface, and exits from the emitting surface. A flow path is formed in the flow path formation. Certain types of antigen capture membranes are exposed inside the channel. The conductor film includes a first main surface and a second main surface. The reflecting surface is in close contact with the first main surface. The specific type of antigen-capturing membrane is fixed on the second main surface.

The sample liquid is filled in the flow path. A specific type of measurement liquid is filled in the flow path before or after the sample liquid is filled in the flow path, and the resonance angle is detected in this state. The specific type of measurement liquid is different from the sample liquid.

After the sample liquid is filled in the flow path and after a specific type of measurement liquid is filled in the flow path and the resonance angle is detected, the flow path is filled with a fluorescent label liquid containing a fluorescent labeled antibody that binds to the antigen. It is. The fluorescent labeling liquid is different from the sample liquid and the specific type of measurement liquid.

  After the fluorescent labeling liquid is filled in the flow path, a specific type of measurement liquid is filled in the flow path, and the amount of surface plasmon excitation fluorescence is measured in that state.

  The second aspect of the present invention adds further matters to the first aspect of the present invention. In the second aspect of the present invention, the difference in the resonance angle due to the difference in the type of the antigen capture film is eliminated by making the refractive indexes different in two or more types of measurement liquids.

  The third aspect of the present invention adds further matters to the second aspect of the present invention. In the third aspect of the present invention, the refractive index is varied by varying the presence or absence of the additive, the type of additive, and the concentration of the additive in all or part of the two or more types of measurement liquids.

  The fourth aspect of the present invention adds a further matter to any one of the first to third aspects of the present invention. In the fourth aspect of the present invention, a sensor chip to which an identifier for identifying the type of the antigen capture membrane is provided is prepared. The identifier is read to identify the type of antigen capture membrane. A specific type of measurement liquid combined with the identified specific type of antigen capture membrane is selected.

  The fifth aspect of the present invention is directed to a measurement apparatus that performs measurement by surface plasmon excitation fluorescence spectroscopy.

  In the fifth aspect of the present invention, a combination storage unit, a sensor chip, a measurement mechanism, an identification mechanism, a liquid feeding mechanism, a measurement liquid selection unit, a primary immune reaction progress unit, a resonance angle detection unit, and a secondary immune reaction progress unit And a measurement unit.

  The combination storage unit stores a combination of two or more types of antigen capture films to which an antibody that binds to an antigen to be measured is fixed and two or more types of measurement liquids and the types of the antigen capture films and the types of the measurement liquids. The combination is determined so that the difference in the resonance angle due to the difference in the type of the antigen capturing film is eliminated.

  The sensor chip includes a dielectric medium, a conductor film, a specific type of antigen capturing film, and a flow path formation. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The incident surface, the reflecting surface, and the emitting surface are arranged so that the excitation light enters the incident surface, is reflected by the reflecting surface, and exits from the emitting surface. A flow path is formed in the flow path formation. Certain types of antigen capture membranes are exposed inside the channel. The conductor film includes a first main surface and a second main surface. The reflecting surface is in close contact with the first main surface. The specific type of antigen-capturing membrane is fixed on the second main surface. The specific type of antigen capture membrane is selected from the two or more types of antigen capture membranes described above.

  The measurement mechanism detects the resonance angle and measures the amount of surface plasmon excitation fluorescence.

  The identification mechanism identifies the type of the antigen capture membrane.

  The measurement liquid selection unit refers to the stored combination, and selects a specific type of measurement liquid combined with the identified specific type of antigen capture membrane.

The liquid feeding mechanism fills the flow path with any of a sample liquid, a fluorescent labeling liquid, and a specific type of measurement liquid. The fluorescent labeling solution contains a fluorescently labeled antibody that binds to the antigen. The sample solution, the fluorescent labeling solution, and the specific type of measurement solution are different from each other.

  The primary immune reaction progression unit controls the liquid feeding mechanism to fill the flow path with the sample liquid.

  The resonance angle detection unit controls the liquid delivery mechanism before or after the sample liquid is filled in the flow path, fills the flow path with a specific type of measurement liquid, controls the measurement mechanism, and controls the specific type. The resonance angle is detected in a state where the measurement liquid is filled in the flow path.

  The secondary immune reaction advancing unit controls the liquid feeding mechanism after the sample liquid is filled in the flow path and after the specific type of measurement liquid is filled in the flow path and the resonance angle is detected. Fill the flow path with liquid.

  The measurement unit controls the liquid delivery mechanism after the fluorescent labeling liquid is filled in the flow path, fills the flow path with a specific type of measurement liquid, controls the measurement mechanism, and measures the amount of surface plasmon excitation fluorescence. Let

  According to the present invention, the detection range of the resonance angle is narrowed, and the resonance angle is easily detected.

  These and other objects, features, aspects and advantages of the invention will become more apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.

It is a schematic diagram of a measuring device. It is a perspective view of a sensor chip. It is a cross-sectional view of a sensor chip. It is a longitudinal cross-sectional view of a sensor chip. It is a schematic diagram of a reagent chip. It is a figure which shows the example of the combination of the kind of antigen capture membrane, and the kind of measurement liquid. It is a graph which shows the relationship between an incident angle and a light quantity. It is a graph which shows the relationship between a density | concentration and a refractive index. It is sectional drawing of the vicinity of the antigen capture | acquisition film | membrane of a sensor chip. It is sectional drawing of the vicinity of the antigen capture | acquisition film | membrane of a sensor chip. It is sectional drawing of the vicinity of the antigen capture | acquisition film | membrane of a sensor chip. It is sectional drawing of the vicinity of the antigen capture | acquisition film | membrane of a sensor chip. It is a top view of a barcode label. It is a block diagram of a controller. It is a flowchart which shows the flow of measurement. It is a flowchart which shows the flow of measurement. It is a schematic diagram of an assembled product.

(Outline)
This desirable embodiment is related with the measuring method and measuring device which measure by surface plasmon excitation fluorescence spectroscopy (SPFS).

(Measuring device components)
The schematic diagram of FIG. 1 shows a measuring device.

  As shown in FIG. 1, the measurement apparatus 1000 includes a main body 1020, a sensor chip 1022, and a reagent chip 1024. The main body 1020 includes a measurement mechanism 1042, a liquid feeding mechanism 1044, a support mechanism 1046, a barcode reader 1048, an operation button 1050, and a controller 1052.

  The measurement mechanism 1042 includes a laser diode 1060, a linear polarizing plate 1062, a mirror 1064, a mirror driving mechanism 1066, a photomultiplier tube 1080, a bandpass filter 1082, a bandpass filter driving mechanism 1084, and a photodiode 1086.

  Components other than these components may be added to the measurement apparatus 1000. Some of these components may be omitted from the measurement apparatus 1000.

(Sensor chip components)
The schematic diagrams from FIG. 2 to FIG. 4 show the sensor chip. 2 to 4 are a perspective view, a transverse sectional view, and a longitudinal sectional view, respectively.

  As shown in FIGS. 2 to 4, the sensor chip 1022 includes a prism 1100, a gold film 1102, an antigen capturing film 1104, a flow path formation 1106, and a barcode label 1108. The flow path forming product 1106 includes a flow path forming sheet 1120 and a flow path forming lid 1122. The prism 1100 includes an incident surface 1140, a reflecting surface 1142, and an exit surface 1144. A flow path 1160 is formed in the flow path formation 1106. An antibody that specifically binds to the antigen to be measured is fixed to the antigen capturing film 1104.

  Components other than these components may be added to the sensor chip 1022.

(Composition of reagent chip)
The schematic diagram of FIG. 5 shows a reagent chip.

  As shown in FIG. 5, the reagent chip 1024 includes a sample container 1180, a dilution container 1182, a dilution liquid container 1184, a fluorescent labeling liquid container 1186, two measurement liquid containers 1188, and a cleaning liquid container 1190. The specimen container 1180 contains the specimen 1210, the dilution container 1182 contains the sample liquid 1212, the dilution liquid container 1184 contains the dilution liquid 1214, and the fluorescent labeling liquid container 1186 contains the fluorescent labeling liquid 1216. Two kinds of measurement liquids 1218 are stored in the two measurement liquid containers 1188, and the cleaning liquid 1220 is stored in the cleaning liquid container 1190. The measurement liquid 1218 is filled in the flow path 1160 when the resonance angle θr is detected and when the light amount of the surface plasmon excitation fluorescence FL is measured.

  Components other than these components may be added to the reagent chip 1024.

(Combination of antigen capture membrane type and measurement solution type)
FIG. 6 shows an example of a combination of the type of antigen capture membrane and the type of measurement solution.

  As shown in FIG. 6, prior to measurement, two types of antigen capture membranes A and B that are candidates for the antigen capture membrane 1104 actually used and two types of candidates for the measurement liquid 1218 that are actually used. Measurement liquids A ′ and B ′ are assumed, and the combination of the type of the antigen capture film 1104 and the type of the measurement liquid 1218 is determined in the two types of antigen capture films A and B and the two types of measurement liquids A ′ and B ′. The For example, the antigen capturing film A and the sample liquid A ′ are combined, and the antigen capturing film B and the sample liquid B ′ are combined.

  When a specific type of antigen capture membrane 1104 that is actually used is selected from the two types of antigen capture membranes A and B, a specific type of measurement liquid 1218 combined with the specific type of antigen capture membrane 1104 is obtained. Selected and actually used. For example, when the sensor chip 1022 including the antigen capturing film A is attached to the main body 1020, the measurement liquid A ′ is filled in the flow path 1160, and when the sensor chip 1022 including the antigen capturing film B is attached to the main body 1020, The measurement liquid B ′ is filled in the flow path 1160.

  The combination of the type of the antigen capture film 1104 and the type of the measurement liquid 1218 is determined so that the difference in the resonance angle θr due to the difference in the type of the antigen capture film 1104 is eliminated. As a result, the detection range of the resonance angle θr is narrowed to the same extent as the range of variations in the resonance angle θr due to other causes, and the resonance angle θr is easily detected.

  Three or more types of antigen capture membrane 1104 candidates may be assumed, and three or more types of measurement liquid 1218 candidates may be assumed. In general, two or more types of antigen capture membranes 1104 that are candidates for the antigen capture membrane 1104 that is actually used and two or more types of measurement solutions 1218 that are candidates for the measurement solution 1218 that are actually used are assumed. In two or more types of antigen capture membranes 1104 and two or more types of measurement solutions 1218, the combination of the type of antigen capture membrane 1104 and the type of measurement solution 1218 is determined.

  When three or more types of antigen capture membrane 1104 candidates are assumed, the number of types of antigen capture membrane 1104 and the number of types of measurement liquid 1218 may be different. For example, the sample liquid A ′ may be combined with the antigen capturing films A and B, and the sample liquid C ′ may be combined with the antigen capturing film C.

  In the two or more types of antigen capture membranes 1104, the type, density, etc. of the immobilized antibody differ depending on the antigen capture membrane 1104. Selection of a specific type of antigen capture membrane 1104 from two or more types of antigen capture membranes 1104 is performed according to the purpose of measurement. For example, a specific type of antigen-capturing membrane 1104 to which an antibody that specifically binds to the antigen is immobilized is selected according to the antigen to be measured. The type of antibody, for example, the molecular weight of the antibody affects the resonance angle θr. The density of the antibody also affects the resonance angle θr. The difference in resonance angle θr depending on the type and density of the antibody may reach about 5 °.

  The resonance angle θr depends on the refractive indices (n and k) of the prism 1100, the gold film 1102, the antigen capturing film 1104, and the measurement liquid 1218. Therefore, the difference in the resonance angle θr due to the difference in the type of the antigen capture film 1104 can be eliminated by making the refractive indexes different in two or more types of measurement liquids 1218.

  Desirably, the refractive index is adjusted by changing the presence or absence of the additive, the type of additive, and the concentration of the additive in all or part of the two or more types of measurement liquids 1218. This facilitates adjustment of the refractive index. However, the refractive index may be adjusted by other than this. For example, the refractive index may be adjusted by changing the type of solvent.

  The additive is preferably sugar, and more preferably sucrose. This facilitates adjustment of the refractive index. In addition, the effect on antibodies, antigens, fluorescently labeled antibodies, etc. is reduced. Furthermore, it becomes easy to obtain additives. In addition, handling of the additive becomes easy. However, the additive may be a sugar other than sucrose and the additive may be other than a sugar as long as the effect on the antibody, antigen, fluorescently labeled antibody, etc. is small.

(Specific examples of combinations)
The graph of FIG. 7 shows the relationship between the incident angle of excitation light and the light quantity of surface plasmon excitation fluorescence. The graph of FIG. 7 shows the relationship between the incident angle of excitation light and the amount of surface plasmon excitation fluorescence for each of two types of sensor chips that differ only in the type of antibody immobilized on the antigen capture membrane. In the measurement of the relationship between the incident angle of excitation light and the amount of surface plasmon excitation fluorescence shown in the graph of FIG. 7, a sample solution having an AFP (α-fetoprotein) antigen concentration extracted from a specimen of 1.0 ng / ml is used. The measurement is performed under the same conditions except for the type of the antigen capture membrane.

  As shown in FIG. 7, in the case of antibody A, the incident angle at which the light amount becomes maximum is 58.4 degrees, and in the case of antibody B, the incident angle at which the light amount becomes maximum is 61.3 degrees. When A and B are compared, a difference of 2.9 degrees is generated in the incident angle at which the light amount is maximized. Thus, even when the antigen is the same and the concentration of the antigen is the same, the resonance angle θr varies depending on the type of the antigen capturing film 1104. Depending on the type of the antigen capturing film 1104, the type of antigen, and the like, a larger difference may occur in the resonance angle θr.

  When the refractive index of the prism 1100 is n = 1.52 and the refractive index of the gold film 1102 is n = 0.26, k = 3.63, the refractive index of the measurement liquid 1218 is only n = 0.01. As it increases, the resonance angle θr increases by 1.58 degrees.

  Therefore, by reducing the refractive index of the measurement liquid combined with the antigen capture membrane on which the antibody B is immobilized by 0.018 less than the refractive index of the measurement liquid combined with the antigen capture membrane on which the antibody A is immobilized, The difference in resonance angle θr due to the difference in type can be eliminated.

  The graph of FIG. 8 shows the relationship between the sucrose concentration and the refractive index.

  As shown in FIG. 8, the higher the sucrose concentration, the greater the refractive index of the measurement liquid 1218. FIG. 8 shows that the refractive index can be increased by 0.018 by increasing the difference in sucrose concentration by about 10%.

(Progress of primary immune response)
When measurement is performed, as illustrated in FIG. 9, the sample liquid 1212 is filled in the flow path 1160 by the liquid feeding mechanism 1044, and the antigen 1242 is captured by the antigen capturing film 1104. The antigen 1242 is captured by a primary immune reaction (antigen-antibody reaction) in which the antibody 1240 immobilized on the antigen capturing film 1104 is bound to the antigen 1242 contained in the sample liquid 1212.

(Resonance angle detection)
After the antigen 1242 is captured, as shown in FIG. 10, the measurement liquid 1218 is filled into the flow path 1160 by the liquid feeding mechanism 1044, and the resonance angle is filled by the measurement mechanism 1042 in a state where the measurement liquid 1218 is filled into the flow path 1160. θr is detected. When the type of the measurement liquid 1218 is selected according to the type of the antigen capture film 1104 so that the difference in the resonance angle θr due to the difference in the type of the antigen capture film 1104 is eliminated, resonance occurs regardless of the type of the antigen capture film 1104. The angle θr is substantially constant. Therefore, the detection range of the resonance angle θr is as narrow as 0 to 1 °, and the detection of the resonance angle θr is easy. This contributes to shortening the time required for detecting the resonance angle θr, reducing the size of the mechanism required for detecting the resonance angle θr, and the like. The type of the antigen capture film 1104 and the type of the measurement liquid 1218 are typically combined so that the resonance angle θr is about 70 °. However, the target of the resonance angle θr is set according to the material and structure of the sensor chip 1022 such as the material and shape of the prism 1100.

(Progress of secondary immune reaction)
After the resonance angle θr is detected, as shown in FIG. 11, the fluorescently labeled liquid 1216 is filled in the flow path 1160 by the liquid feeding mechanism 1044, and the fluorescently labeled antibody 1244 is added to the antigen 1242 captured by the antigen capturing film 1104. Is done. The fluorescently labeled antibody 1244 is added by a secondary immune reaction (antigen-antibody reaction) in which the antigen 1242 captured by the antigen capturing film 1104 and the fluorescently labeled antibody 1244 contained in the fluorescent labeling liquid 1216 are bound.

(Measurement of light intensity of surface plasmon excitation fluorescence)
After the fluorescently labeled antibody 1244 is added, as shown in FIG. 12, the measurement liquid 1218 is filled in the flow path 1160 by the liquid feeding mechanism 1044, and the measurement mechanism 1042 is filled with the measurement liquid 1218 in the flow path 1160. The amount of the surface plasmon excitation fluorescence FL is measured.

(Structure of sensor chip)
As shown in FIGS. 2 to 4, the reflecting surface 1142 is brought into close contact with one main surface 1260 of the gold film 1102, and the antigen capturing film 1104 is fixed to the other main surface 1262 of the gold film 1102. The flow path formation 1106 is bonded to the prism 1100 on which the gold film 1102 and the antigen capturing film 1104 are formed.

  The sensor chip 1022 is also called “inspection chip”, “analysis chip”, “biochip”, “sample cell”, or the like. The sensor chip 1022 is desirably a structure in which the length of each side is in the range of several millimeters to several centimeters, but may be a smaller or larger structure.

(prism)
As shown in FIGS. 2 to 4, the prism 1100 is a trapezoidal column. The prism 1100 is preferably an isosceles trapezoidal column. One inclined side surface of the prism 1100 becomes an incident surface 1140. The wide parallel side surface of the prism 1100 becomes a reflection surface 1142. The other inclined side surface of the prism 1100 becomes an exit surface 1144.

  The incident surface 1140, the reflecting surface 1142, and the emitting surface 1144 are arranged so that the excitation light EL enters the incident surface 1140, is reflected by the reflecting surface 1142, and is emitted from the emitting surface 1144. The excitation light EL satisfies the total reflection condition and enters the reflection surface 1142.

  The shape of the prism 1100 is determined so that the excitation light EL can be incident on the reflecting surface 1142 at the resonance angle θr. As long as this condition is satisfied, the prism 1100 may be other than the trapezoidal column, and the prism 1100 may be replaced with a shape that is difficult to call a “prism”. For example, the prism 1100 may be a semi-cylindrical body, and the prism 1100 may be replaced with a plate.

  The prism 1100 is a dielectric medium made of a dielectric that is transparent to the excitation light EL. The material of the prism 1100 is resin, glass, or the like. When the material of the prism 1100 is resin, the prism 1100 is desirably manufactured by injection molding.

(Gold film)
The gold film 1102 is a thin film. The film thickness of the gold film 1102 is desirably 100 nm or less, and more desirably 40 to 50 nm. However, the film thickness of the gold film 1102 may be outside this range.

  The gold film 1102 may be replaced with a conductor film made of another type of conductor that generates surface plasmon resonance. For example, the gold film 1102 may be replaced with a conductor film made of a metal such as silver, copper, or aluminum, or an alloy containing these metals.

  The gold film 1102 is formed by sputtering, vapor deposition, plating, or the like.

(Antigen capture membrane)
As shown in FIGS. 3 and 4, the antigen capturing film 1104 is exposed inside the flow path 1160. Therefore, when the liquid is filled in the flow path 1160, the liquid filled in the flow path 1160 comes into contact with the antigen capturing film 1104. When the sample liquid 1212 containing the antigen 1242 to be measured comes into contact with the antigen capturing film 1104, the antibody 1240 fixed to the antigen capturing film 1104 and the antigen 1242 included in the sample liquid 1212 are combined. In the state where the antigen 1242 to be measured is captured by the antigen capture film 1104, when the fluorescent labeling solution 1216 containing the fluorescently labeled antibody 1244 comes into contact with the antigen capture film 1104, the antigen 1242 captured by the antigen capture film 1104 and the fluorescence The fluorescently labeled antibody 1244 contained in the labeling liquid 1216 is bound.

  The antigen capturing film 1104 is made of a non-fluid. Therefore, even if the liquid contacts the antigen capturing film 1104, the antigen capturing film 1104 does not move.

  The antigen capturing film 1104 is patterned by a rubber applicator and fixed only on a part of the other main surface 1262 of the gold film 1102. The antigen capturing film 1104 may be patterned by other methods. The planar shape of the antigen capturing film 1104 is a circle, a polygon, or the like. The diameter of the antigen capturing membrane 1104 is desirably several mm. The thickness of the antigen capturing film 1104 is desirably 100 nm or less, and more desirably 60 nm or less.

  When the antigen capturing film 1104 is formed, a self-assembled (SAM) film is formed on the other main surface 1262 of the gold film 1102, a solid support layer is formed on the self-assembled film, and the solid support Antibody 1240 is immobilized on the body layer.

  When the self-assembled film is formed, the prism 1100 in which the gold film 1102 is formed is immersed in a liquid in which a substance constituting the self-assembled film is dissolved or dispersed. For example, a prism 1100 on which a gold film 1102 is formed is immersed in an ethanol solution of an alkanethiol derivative such as 11-amino-1-undecanethiol.

  When the solid support layer is formed, the prism 1100 on which the gold film 1102 is formed is immersed in a liquid in which a substance constituting the solid support layer is dissolved or dispersed. For example, the prism 1100 on which the gold film 1102 is formed is immersed in a buffer solution in which carboxymethyl dextran (CMD) is dissolved. The buffer solution is, for example, an aqueous solution of 2-morpholinoethanesulfonic acid (MES) and sodium chloride (NaCl).

(Channel formation)
As shown in FIG. 4, the flow path 1160 reaches from the one opening 1300 to the other opening 1302 via the inside of the flow path formation 1106. A part of the channel 1160 is exposed to the joint surface.

  A reaction chamber 1322 is formed in the flow path forming sheet 1120. Paths 1340 and 1342 are formed in the flow path forming lid 1122. A flow path 1160 is constituted by the reaction chamber 1322 and the paths 1340 and 1342.

  The reaction chamber 1322 is a hole that penetrates both main surfaces of the flow path forming sheet 1120. One path 1340 is a hole extending from one opening 1300 to one end of the reaction chamber 1322. The other path 1342 is a hole extending from the other end of the reaction chamber 1322 to the other opening 1302.

  The flow path forming sheet 1120 is a double-sided pressure-sensitive adhesive sheet in which layers made of a pressure-sensitive adhesive such as an acrylic pressure-sensitive adhesive are formed on both main surfaces of a base material made of polyethylene terephthalate or the like, and the gold film 1102 and the antigen capturing film 1104 are The formed prism 1100 and the flow path forming lid 1122 are joined.

  The flow path forming lid 1122 is made of a material transparent to the surface plasmon excitation fluorescence FL. The flow path forming lid 1122 is made of, for example, resin or glass. When the flow path forming lid 1122 is made of resin, the flow path forming lid 1122 is desirably manufactured by injection molding.

  The flow path forming sheet 1120 and the flow path forming lid 1122 may be integrated.

(Barcode label)
The schematic diagram of FIG. 13 is a plan view of the barcode label.

  As shown in FIG. 13, a barcode 1360 is drawn on the barcode label 1108. The bar code label 1108 is attached to the component of the sensor chip 1022. The bar code label 1108 is affixed to a place where the optical paths of the excitation light EL and the surface plasmon excitation fluorescence FL are not blocked and the openings 1300 and 1302 are not blocked.

  The barcode 1360 serves as an identifier for identifying the type of the antigen capturing film 1104. Bar code 1360 may be replaced with other types of identifiers. For example, the barcode 1360 may be replaced with a hologram, a two-dimensional code, characters, symbols, and the like.

  The identifier may be attached to the sensor chip 1022 by a method other than attaching the label to the component. For example, the identifier may be drawn directly on the component of the sensor chip 1022. A structure such as a notch serving as an identifier may be formed in the component of the sensor chip 1022.

(Barcode reader)
The barcode reader 1048 reads the barcode 1360 with an image sensor and identifies the type of the antigen capturing film 1104. When the barcode 1360 is replaced with another type of identifier, an identification mechanism suitable for identifying the other type of identifier is provided. For example, when a structure such as a notch is formed as an identifier, a limit switch, an optical sensor, or the like that detects the structure is provided.

(Support mechanism)
As shown in FIG. 1, the support mechanism 1046 supports the sensor chip 1022 and positions the sensor chip 1022 at a certain position. The sensor chip 1022 can be attached to and detached from the support mechanism 1046, and is exchanged for each measurement. The type of the antigen capture film 1104 provided in the sensor chip 1022 supported by the support mechanism 1046 may be different for each measurement. However, in the measuring apparatus 1000, the type of the measurement liquid 1218 is selected according to the type of the antigen capturing film 1104, and the resonance angle θr becomes substantially constant even if the type of the antigen capturing film 1104 is different.

(Liquid feeding mechanism)
As shown in FIG. 1, the liquid feeding mechanism 1044 supplies the liquid from the reagent chip 1024 to the sensor chip 1022 and collects the liquid from the sensor chip 1022. When the liquid is supplied to the sensor chip 1022, the liquid is supplied to one opening 1300, the channel 1160 is filled with the liquid, and the liquid contacts the antigen capturing film 1104. When the liquid is recovered from the sensor chip 1022, the liquid is recovered from one opening 1300, and the flow path 1160 becomes empty or nearly empty.

  For example, the liquid feeding mechanism 1044 sucks the liquid from the liquid feeding source by a pump, conveys the pump from the liquid feeding source to the liquid feeding destination, and discharges the liquid to the liquid feeding destination by the pump. The liquid may flow through a pipe from the liquid supply source to the liquid supply destination.

(Laser diode)
As shown in FIG. 1, the laser diode 1060 emits excitation light EL.

  The laser diode 1060 may be replaced with other types of light sources. For example, the laser diode 1060 may be replaced with a light emitting diode, a mercury lamp, a laser other than the laser diode, or the like.

  When the light emitted from the light source is not a parallel light beam, the light is converted into a parallel light beam by a lens, a mirror, a slit, or the like. When the light is not linearly polarized light, the light is converted into linearly polarized light by a linear polarizing plate or the like. When the light is not monochromatic light, the light is converted into monochromatic light by a diffraction grating or the like.

(Linear polarizing plate)
As shown in FIG. 1, the linearly polarizing plate 1062 is disposed on the optical path of the excitation light EL, and converts the excitation light EL emitted from the laser diode 1060 into linearly polarized light. The polarization direction of the excitation light EL is selected so that the excitation light EL is p-polarized with respect to the reflection surface 1142 of the prism 1100. As a result, leakage of the evanescent wave increases, the amount of light of the surface plasmon excitation fluorescence FL increases, and measurement accuracy and sensitivity are improved.

(Mirror and mirror drive mechanism)
As shown in FIG. 1, the mirror 1064 is disposed on the optical path of the excitation light EL, and reflects the excitation light EL that has passed through the linearly polarizing plate 1062. The excitation light EL reflected by the mirror 1064 is applied to the prism 1100. As shown in FIG. 3, the light applied to the prism 1100 enters the incident surface 1140, is reflected by the reflecting surface 1142, and exits from the exit surface 1144. The incident angle θ of the excitation light EL to the reflecting surface 1142 is larger than the critical angle θc.

  The mirror driving mechanism 1066 includes a driving force source such as a piezoelectric actuator and an electromagnetic motor, and rotates the mirror 1064 to adjust the posture of the mirror 1064. The mirror driving mechanism 1066 includes a driving force source such as a linear stepping motor, and moves the mirror 1064 in the optical axis direction of the laser diode 1060 to adjust the position of the mirror 1064. Thereby, the incident angle θ of the excitation light EL to the reflection surface 1142 can be adjusted while maintaining the incident position of the excitation light EL on the reflection surface 1142 on the back side of the region where the antigen capturing film 1104 is fixed.

  The incident angle θ may be adjusted by adjusting the position or posture of a component other than the mirror 1064 of the measurement mechanism 1042 or the sensor chip 1022.

(Photomultiplier tube)
As shown in FIG. 1, the photomultiplier tube 1080 is disposed on the optical path of the surface plasmon excitation fluorescence FL emitted from the sensor chip 1022, and measures the amount of light of the surface plasmon excitation fluorescence FL. The photomultiplier tube 1080 may be replaced with another type of light quantity sensor. For example, the photomultiplier tube 1080 may be replaced with a charge coupled device (CCD) sensor or the like.

(Band pass filter)
As shown in FIG. 1, the bandpass filter 1082 is disposed on the optical path of the surface plasmon excitation fluorescence FL. The band pass filter 1082 transmits light having a wavelength included in the pass band and attenuates light having a wavelength included in the stop band. The specifications of the bandpass filter 1082 are selected so that the pass band includes the wavelength of the surface plasmon excitation fluorescence FL and the stop band includes the wavelength of the excitation light EL.

  Scattered excitation light EL (hereinafter referred to as “scattered light”) is attenuated by the bandpass filter 1082 and only a part of the scattered light reaches the photomultiplier tube 1080, but the surface plasmon excitation fluorescence FL is bandpass. Most of the surface plasmon excitation fluorescence FL passes through the filter 1082 and reaches the photomultiplier tube 1080. Thereby, when the light quantity of the relatively small surface plasmon excitation fluorescence FL is measured, the influence of a scattered light is suppressed and the measurement precision improves. The band pass filter 1082 may be replaced with a low pass filter.

(Band pass filter drive mechanism)
As shown in FIG. 1, the bandpass filter drive mechanism 1084 includes a state in which the bandpass filter 1082 is disposed on the optical path of the surface plasmon excitation fluorescence FL and a bandpass filter 1082 that is not disposed on the optical path of the surface plasmon excitation fluorescence FL. Switch between states.

(Photodiode)
As shown in FIG. 1, the photodiode 1086 is disposed on the optical path of the excitation light EL (hereinafter referred to as “reflected light”) reflected at the interface between the prism 1100 and the gold film 1102 and measures the amount of reflected light. . The photodiode 1086 may be replaced with another type of light quantity sensor. For example, the photodiode 1086 may be replaced with a phototransistor, a photoresistor, or the like. If the resonance angle θr is detected regardless of the amount of reflected light, the photodiode 1086 may be omitted.

(Reagent chip)
The reagent chip 1024 is provided to the operator in a state in which a diluent 1214, a fluorescent labeling liquid 1216, two kinds of measurement liquids 1218, and a cleaning liquid 1220 are accommodated. All or a part of the diluent 1214, the fluorescent labeling liquid 1216, the two types of measurement liquid 1218, and the cleaning liquid 1220 may be accommodated in the reagent chip 1024 by the operator. The specimen 1210 is accommodated in the reagent chip 1024 by the operator. The sample liquid 1212 is prepared from the specimen 1210 and the diluted liquid 1214 inside the measuring apparatus 1000. Two types of measurement liquid 1218 may be prepared by an operator or inside the measurement apparatus 1000.

(Sample)
The specimen 1210 is typically a human sample such as blood, serum, plasma, urine, nasal fluid, saliva, body cavity fluid, etc., but it may be a sample from a non-human organism. It may also be a harvest from a living organism. The body cavity fluid includes cerebrospinal fluid, ascites, pleural effusion and the like.

(Measurement solution)
The measurement solution 1218 is a buffer solution having a buffer capacity for maintaining a pH at which the antibody 1240, the antigen 1242, and the fluorescently labeled antibody 1244 are not inactivated. As long as the antibody 1240, the antigen 1242, and the fluorescently labeled antibody 1244 are not inactivated and measurement of the amount of light of the surface plasmon excitation fluorescence FL is not inhibited, a solution having no buffering capacity may be used instead of the buffer solution. Desirably, the temperature of the measurement liquid 1218 is kept constant.

(Fluorescent labeling solution)
The fluorescent labeling liquid 1216 includes a fluorescently labeled antibody 1244 that specifically binds to the antigen 1242 to be measured. A fluorescent label that is excited by an electric field of evanescent waves is added to the fluorescently labeled antibody 1244.

(Cleaning solution)
Desirably, a surfactant is added to the cleaning liquid 1220.

(Antigen to be measured)
The antigen 1242 to be measured may be a nucleic acid, protein, amino acid, carbohydrate, lipid, or the like, or may be a modified molecule obtained by modifying these, and two or more of these complexes It may be a body. The nucleic acid may be single-stranded or double-stranded. The nucleic acid may be DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., may be a nucleoside, nucleotide or the like, and is a modified molecule obtained by modifying a nucleoside, nucleotide or the like. It may be a complex of two or more of nucleosides and nucleotides. Examples of proteins include polypeptides and oligopeptides. Amino acids also include modified amino acids. Specifically, the antigen 1242 to be measured is a carcinoembryonic antigen such as AFP or a tumor marker, a signaling substance, a hormone, or the like, but may be other than these.

(controller)
The controller 1052 is an embedded computer that executes a control program. One embedded computer may be responsible for the function of the controller 1052, or two or more embedded computers may be responsible for the function of the controller 1052. The embedded computer is provided with a CPU and a memory. Hardware without software may be responsible for all or part of the functions of the controller 1052. The hardware is, for example, an electronic circuit such as an operational amplifier or a comparator, but a mechanical mechanism such as a link mechanism may be included in the hardware. All or part of the processing by the controller 1052 may be executed manually or may be executed outside the measuring apparatus 1000. The controller 1052 controls components of the measurement device 1000 such as the measurement mechanism 1042, the liquid feeding mechanism 1044, and the barcode reader 1048.

  The block diagram of FIG. 14 shows the controller.

  As shown in FIG. 14, the controller 1052 includes a sample solution preparation unit 1380, a combination storage unit 1382, a measurement solution selection unit 1386, a primary immune reaction progress unit 1388, a cleaning unit 1390, a resonance angle detection unit 1392, and a secondary immune reaction. A progression unit 1394 and a measurement unit 1396 are provided. The combination storage unit 1382 stores a combination of the type of the antigen capturing film 1104 and the type of the measurement liquid 1218. The contents of control by blocks other than the combination storage unit 1382 will be described in the column of measurement flow. The controller 1052 may perform control other than the control by these blocks.

(Measurement flow)
The flowcharts of FIGS. 15 and 16 show the flow of measurement.

(Determination of the combination)
As shown in FIGS. 15 and 16, prior to the main measurement, a combination of the type of the antigen capture film 1104 and the type of the measurement liquid 1218 is determined (step S100). The determined combination of the type of the antigen capturing film 1104 and the type of the measurement liquid 1218 is input to the main body 1000 and stored in the combination storage unit 1382. The combination of the type of the antigen capturing film 1104 and the type of the measurement liquid 1218 may be experimentally determined by prior measurement or may be theoretically determined. The determination of the combination of the type of the antigen capture film 1104 and the type of the measurement liquid 1218 need not be performed for each measurement. The determined combination of the type of the antigen capture film 1104 and the type of the measurement liquid 1218 is repeatedly referred to in two or more measurements.

  When measurement is performed, a specific type of antigen capturing film 1104 to which an antibody that specifically binds to the antigen to be measured is fixed is selected (step S101).

(Preparation of main body, sensor chip and reagent chip)
After the specific type of antigen capture film 1104 is selected, the main body 1020, the sensor chip 1022, and the reagent chip 1024 are prepared (step S102). The main body 1020 is provided in a state where the combination of the type of the antigen capturing film 1104 and the type of the measurement liquid 1218 is stored in the combination storage unit 1382. The sensor chip 1022 is selected to include the selected specific type of antigen capture film 1104.

(Attaching the sensor chip and reagent chip)
After the main body 1020, the sensor chip 1022, and the reagent chip 1024 are prepared, the sensor chip 1022 and the reagent chip 1024 are attached to the main body 1020 (step S103).

(Preparation of sample solution)
After the sensor chip 1022 and the reagent chip 1024 are attached, the sample liquid 1212 is prepared (step S104). When the sample liquid 1212 is prepared, the sample liquid preparation unit 1380 controls the liquid feeding mechanism 1044, and the specimen 1210 and the diluted liquid 1214 are mixed by the liquid feeding mechanism 1044. The specimen 1210 may be used as the sample liquid 1212 as it is. The sample solution 1212 may be prepared manually. When the sample solution 1212 is prepared manually, the manual operation may be performed inside the measuring apparatus 1000 or may be performed outside. The prepared sample solution 1212 is stored in the dilution container 1182. When the sample solution 1212 is prepared, pretreatment other than dilution may be performed. For example, pretreatment such as blood cell separation or reagent mixing may be performed.

(Identification of antigen capture membrane type)
After the sensor chip 1022 and the reagent chip 1024 are attached, the barcode 1360 is read by the barcode reader 1048, and the type of the antigen capture film 1104 is identified (step S105). The operation button 1050 may accept an input of the type of the antigen capture film 1104, and the type of the antigen capture film 1104 may be identified from the input result. The operation button 1050 may be replaced with another type of input device. For example, the operation button 1050 may be replaced with a keyboard, a touch panel, or the like. When the measurement liquid 1218 is selected by an operator's manual operation, identification of the type of the antigen capture film 1104 by the barcode reader 1048 may be omitted.

(Selection of measurement solution)
After the type of the antigen capture film 1104 is identified, the measurement liquid selection unit 1386 refers to the combination stored in the combination storage unit 1382 and combines it with the specific type of antigen capture film 1104 identified by the barcode reader 1048. The specified type of measurement liquid 1218 is selected (step S106). The two types of measurement liquids 1218 do not need to be prepared in advance, and a specific type of measurement liquid 1218 combined with a specific type of antigen capture film 1104 may be prepared for each measurement.

(Progress of primary immune response)
After the sample liquid 1212 is prepared, the primary immune reaction progressing unit 1388 controls the liquid feeding mechanism 1044, and the liquid feeding mechanism 1044 supplies the sample liquid 1212 to the sensor chip 1022 and collects it from the sensor chip 1022. The reaction is allowed to proceed (step S107). The sample liquid 1212 is filled in the channel 1160 from when the sample liquid 1212 is supplied to the sensor chip 1022 until it is recovered from the sensor chip 1022. As a result, the sample liquid 1212 comes into contact with the antigen capturing film 1104, and the antigen 1242 contained in the sample liquid 1212 is captured by the antigen capturing film 1104. The sample solution 1212 may be filled in the flow path 1160 to such an extent that the sample solution 1212 is sufficiently in contact with the antigen capturing film 1104. That is, it is only necessary that the reaction chamber 1322 is mainly filled with the sample solution 1212, and it is not always necessary that the entire channel 1160 is filled with the sample solution 1212.

  The primary immune reaction is allowed to proceed before or after the identification of the type of the antigen capture membrane 1104 and the selection of the measurement liquid 1218 or in parallel with the identification of the type of the antigen capture film 1104 and the selection of the measurement liquid 1218.

(Flow path cleaning)
After the primary immune reaction, the washing unit 1390 controls the liquid feeding mechanism 1044, the washing liquid 1220 is supplied to the sensor chip 1022 by the liquid feeding mechanism 1044 and collected from the sensor chip 1022, and the flow path 1160 is washed (step S108). ). As a result, unreacted antigen 1242, unnecessary substances, and the like are removed from the flow path 1160. When the amount of unreacted antigen 1242 and unwanted matter remaining is negligibly small, the cleaning of the flow path 1160 may be omitted.

(Resonance angle detection)
After the flow path 1160 is washed, the resonance angle detector 1392 controls the measurement mechanism 1042 and the liquid feeding mechanism 1044, and the resonance angle is detected (step S109).

  When the resonance angle is detected, the selected specific type of measurement liquid 1218 is supplied to the sensor chip 1022 by the liquid feeding mechanism 1044 and collected from the sensor chip 1022. The measurement liquid 1218 is filled in the flow path 1160 until the measurement liquid 1218 is supplied to the sensor chip and then collected from the sensor chip. The measurement liquid 1218 may be filled in the flow path 1160 to such an extent that it fills the space where the evanescent wave leaks.

  In a state where the flow path 1160 is filled with the measurement liquid 1218, the excitation light EL is irradiated to the prism 1100 by the measurement mechanism 1042. The excitation light EL is incident on the incident surface 1140, satisfies the total reflection condition, is incident on the reflective surface 1142, is reflected by the reflective surface 1142, and is reflected from the output surface 1144. At this time, the incident angle θ is scanned in the vicinity of the resonance angle θr expected by the mirror driving mechanism 1066. While the incident angle θ is scanned, the amount of reflected light is measured by the photodiode 1086. The incident angle θ at which the amount of reflected light is minimized or its correction value is regarded as the resonance angle θr at which the electric field enhancement is maximized. If the difference between the incident angle θ at which the amount of reflected light is minimized and the incident angle θ at which the electric field enhancement is at a maximum cannot be ignored, a small angle is added to or subtracted from the incident angle θ at which the amount of reflected light is minimized. Correction is performed. The resonance angle θr may be detected by other methods. For example, the resonance angle θr may be detected from the incident angle θ at which the amount of scattered light is maximized. The amount of scattered light may be measured by the photomultiplier tube 1080 or may be measured by a light amount sensor other than the photomultiplier tube 1080 and the photodiode 1086. When the amount of scattered light is measured by the photomultiplier tube 1080, the bandpass filter 1082 is retracted from the optical path of the surface plasmon excitation fluorescence FL by the bandpass filter driving mechanism 1084.

  When the resonance angle θr is detected after the primary immune reaction, the influence of the change in the resonance angle θr due to the capture of the antigen 1242 is eliminated, and the measurement accuracy and sensitivity are improved. However, the resonance angle θr may be detected before the primary immune reaction.

(Progress of secondary immune reaction)
After the resonance angle θr is detected, the secondary immune reaction progressing unit 1394 controls the liquid feeding mechanism 1044, and the fluorescent labeling liquid 1216 is supplied to the sensor chip 1022 by the liquid feeding mechanism 1044 and collected from the sensor chip 1022, and the secondary An immune reaction is allowed to proceed (step S110). The fluorescent labeling liquid 1216 is filled in the flow path 1160 from when the fluorescent labeling liquid 1216 is supplied to the sensor chip 1022 until it is collected. As a result, the fluorescent labeling liquid 1216 comes into contact with the antigen capturing film 1104, and the fluorescently labeled antibody 1244 is added to the antigen 1242 captured by the antigen capturing film 1104.

(Flow path cleaning)
After the secondary immune reaction, the washing unit 1390 controls the liquid feeding mechanism 1044, the washing liquid 1220 is supplied to the sensor chip 1022 by the liquid feeding mechanism 1044 and collected from the sensor chip 1022, and the flow path 1160 is washed (step S111). ). As a result, unreacted fluorescently labeled antibody 1244, unnecessary substances and the like are removed from the flow path 1160. When the unreacted fluorescence-labeled antibody 1244 and the amount of unwanted matter remaining are negligibly small, the washing of the flow path 1160 may be omitted.

(Measurement)
After the flow path 1160 is washed, the measurement unit 1396 controls the measurement mechanism 1042 and the liquid feeding mechanism 1044, and the light amount of the surface plasmon excitation fluorescence FL is measured (step S112).

When the amount of light of the surface plasmon excitation fluorescence FL is measured, the measurement liquid 1218 is supplied to the sensor chip 1022 by the liquid feeding mechanism 1044 and collected from the sensor chip 1022. Between the time when the measurement liquid 1218 is supplied to the sensor chip 1022 and the time when the measurement liquid 1218 is collected from the sensor chip 1022, the measurement liquid 1218 is filled in the flow path 1160. The measurement liquid 1218 may be filled in the flow path 1160 to such an extent that it fills the space where the evanescent wave leaks.

  In a state where the flow path 1160 is filled with the measurement liquid 1218, the excitation light EL is irradiated to the prism 1100 by the measurement mechanism 1042. The excitation light EL enters the incident surface 1140, enters the reflecting surface 1142 at the resonance angle θr, is reflected by the reflecting surface 1142, and exits from the emitting surface 1144. The incident angle of the excitation light EL to the reflecting surface 1142 is set to the resonance angle θr. At this time, as shown in FIG. 12, the evanescent wave EW leaks from the interface between the prism 1100 and the gold film 1102 to the gold film 1102, and the evanescent wave EW and the plasmon on the surface of the gold film 1102 resonate. The electric field of the evanescent wave EW is enhanced. The fluorescence labeled antibody 1244 added to the antigen 1242 captured by the antigen capture film 1104 is excited by the electric field of the enhanced evanescent wave EW, and the fluorescence labeled antibody 1244 added to the antigen 1242 captured by the antigen capture film 1104 Surface plasmon excitation fluorescence FL is emitted. While the excitation light EL is irradiated, the light quantity of the surface plasmon excitation fluorescence FL emitted from the sensor chip 1022 is measured by the photomultiplier tube 1080. The measured light amount of the surface plasmon excitation fluorescence FL is used to specify the amount of antigen 1242 captured, the presence or absence of antigen 1242 capture, and the like.

(Provide assembled products)
The reagent chip 1024 shown in FIG. 5 contains two types of measurement liquids 1218. In the measurement apparatus 1000, two types of measurement liquids are used depending on the type of the antigen capture film 1104 provided in the sensor chip 1022 held by the support mechanism 1046. A specific type of measurement liquid 1218 is selected from 1218.

  However, only one type of measurement liquid can be stored in the reagent chip. For example, as shown in the schematic diagram of FIG. 17, an assembled product 1410 in which a sensor chip 1022 and a reagent chip 1024a are paired is provided, and only one type of antigen capture film 1104 and one type of reagent chip 1024a of the sensor chip 1022 are provided. When the type of the measurement liquid 1218 to be provided is selected according to the above-described combination, the sensor chip 1022 and the reagent chip 1024a that make a pair are attached to the measurement apparatus 1000, thereby identifying the type of the antigen capture film and the sample liquid. Even if this selection is not performed, the resonance angle θr can be made substantially constant. In order to make the sensor chip 1022 and the reagent chip 1024a into a combined product, the sensor chip 1022 and the reagent chip 1024a may be packaged integrally, or the sensor chip 1022 and the reagent chip 1024a are paired. The identifier may be given to the sensor chip 1022 and the reagent chip 1024a.

  Although the present invention has been described in detail, the above description is illustrative in all aspects, and the present invention is not limited to the above description. Innumerable modifications not illustrated can be envisaged without departing from the scope of the present invention.

1022 Sensor chip 1042 Measuring mechanism 1044 Liquid feeding mechanism 1048 Barcode reader 1104 Antigen capturing film 1160 Channel 1212 Sample liquid 1216 Fluorescent labeling liquid 1218 Measuring liquid

Claims (5)

A measurement method for measuring by surface plasmon excitation fluorescence spectroscopy,
(a) determining a combination of the type of the antigen capture membrane and the type of the measurement solution in two or more types of the antigen capture membrane and two or more types of measurement solutions to which an antibody that binds to the antigen to be measured is fixed;
(b) selecting a specific type of antigen capture membrane from the two or more types of antigen capture membranes;
(c) A dielectric medium, a conductor film, a specific type of antigen capturing film, and a flow path formation, the dielectric medium having an incident surface, a reflective surface, and an exit surface, and excitation light is incident on the incident surface The incident surface, the reflection surface, and the emission surface are arranged so as to be reflected by the reflection surface and emitted from the emission surface, and a flow channel is formed in the flow channel formation object. Is exposed to the inside of the flow path, the conductor film has a first main surface and a second main surface, the reflective surface is in close contact with the first main surface, and captures the specific type of antigen Preparing a sensor chip for fixing a film on the second main surface;
(d) selecting a specific type of measurement liquid combined with the specific type of antigen capture membrane in the combination;
(e) filling the sample liquid into the flow path;
(f) Before or after the step (e), the specific type of measurement liquid different from the sample liquid is filled in the flow path, and the resonance angle in a state where the specific type of measurement liquid is filled in the flow path. Detecting
(g) after the step (e) and the step (f), the step of filling the flow path with a fluorescently labeled solution containing a fluorescently labeled antibody that binds to the antigen and different from the sample solution and the specific type of measuring solution ; ,
(h) After the step (g), the specific type of measurement liquid is filled in the flow path, and the amount of surface plasmon excitation fluorescence is measured in a state where the specific type of measurement liquid is filled in the flow path. Process,
With
The step (a)
A measurement method for determining a combination of a type of an antigen capture film and a type of a measurement solution so as to eliminate the difference in the resonance angle due to the difference in the type of the antigen capture film. In the measuring method of Claim 1,
The step (a)
A measurement method that eliminates the difference in the resonance angle due to the difference in the type of the antigen-capturing film by changing the refractive index in the two or more types of measurement liquids. In the measuring method of Claim 2,
The step (a)
A measurement method in which the refractive index is varied by varying the presence or absence of an additive, the type of additive, and the concentration of the additive in all or part of the two or more types of measurement solutions. In the measuring method in any one of Claim 1 to Claim 3,
The step (c)
Preparing the sensor chip to which an identifier for identifying the type of the antigen-capturing membrane is attached;
(i) reading the identifier and identifying the type of the antigen capture membrane;
Further comprising
The step (d)
A measurement method for selecting a specific type of measurement liquid combined with the specific type of antigen-capturing membrane identified in the step (i). A measuring device for measuring by surface plasmon excitation fluorescence spectroscopy,
A combination storage unit that stores a combination of two or more types of antigen capture membranes to which an antibody that binds to an antigen to be measured is fixed, and two or more types of measurement solutions, and the types of antigen capture membranes and measurement solutions;
A dielectric medium, a conductor film, a specific type of antigen capture film, and a flow path formation, wherein the dielectric medium has an incident surface, a reflective surface, and an output surface, and excitation light is incident on the incident surface and reflected. The incident surface, the reflective surface, and the exit surface are arranged so as to be reflected by a surface and exit from the exit surface, and the specific type of antigen capture film is selected from the two or more types of antigen capture films, and the flow A flow path is formed in the path formation, the specific type of antigen capturing film is exposed inside the flow path, the conductor film includes a first main surface and a second main surface, and the reflective surface A sensor chip that adheres to the first main surface, and the specific type of antigen-capturing film is fixed to the second main surface;
A measurement mechanism for detecting the resonance angle and measuring the amount of surface plasmon excitation fluorescence;
An identification mechanism for identifying the type of antigen capture membrane;
A measurement liquid selection unit that refers to the combination and selects a specific type of measurement liquid combined with the specific type of antigen capture membrane identified by the identification mechanism;
A liquid feeding mechanism that fills the flow path with different sample liquids, a fluorescently labeled liquid containing a fluorescently labeled antibody that binds to the antigen, and the specific type of measuring liquid;
A primary immune reaction progressing part that controls the liquid feeding mechanism and fills the flow path with the sample liquid;
Before or after the sample liquid is filled in the flow path, the liquid feeding mechanism is controlled, the specific type of measurement liquid is filled in the flow path, the measurement mechanism is controlled, and the specific liquid is A resonance angle detection unit that detects the resonance angle in a state where the type of measurement liquid is filled in the flow path;
After the sample liquid is filled in the flow path and after the specific type of measurement liquid is filled in the flow path and the resonance angle is detected, the liquid feeding mechanism is controlled, and the fluorescent labeling liquid A secondary immune reaction progressing section that fills the flow path,
After the fluorescent labeling liquid is filled in the flow path, the liquid feeding mechanism is controlled, the specific type of measurement liquid is filled in the flow path, the measurement mechanism is controlled, and the surface plasmon excitation fluorescence is controlled. A measurement unit for measuring the amount of light;
With
The combination storage unit
A measuring apparatus for storing a combination of a type of an antigen capturing film and a type of a measuring solution that eliminates the difference in the resonance angle due to the difference in the type of the antigen capturing film.

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