JP4864743B2 - Fluorescence measuring device - Google Patents

Description

  The present invention relates to a fluorescence measuring apparatus for detecting a specific substance in a sample by a fluorescence method, and more particularly to a fluorescence measuring apparatus of a type in which a sensor unit is immersed in a sample solution.

  Conventionally, a fluorescence method has been widely used as a highly sensitive and easy measurement method in biomeasurement and the like. In this fluorescence method, a sample that is considered to contain a measurement target substance that emits fluorescence when excited by light of a specific wavelength is irradiated with the excitation light of the specific wavelength, and then the presence of the measurement target substance is detected by detecting the fluorescence. It is a method to confirm. In addition, when the measurement target substance is not a phosphor, this binding, that is, by detecting the fluorescence in the same manner as described above, by contacting the sample with a substance that is labeled with the fluorescent substance and specifically binds to the measurement target substance. Confirmation of the existence of a substance to be measured is also widely performed.

  FIG. 10 schematically shows an example of a sensor that performs the fluorescence method using the labeled substance. The fluorescence measuring apparatus of this example is for detecting the antigen 2 contained in the liquid sample 1 as an example, and the substrate 3 is coated with a primary antibody 4 that specifically binds to the antigen 2. Then, the liquid sample 1 is caused to flow in the sample holder 5 provided on the substrate 3, and then the secondary antibody 6 that is similarly labeled with the phosphor 10 and specifically binds to the antigen 2 is caused to flow. Thereafter, excitation light 8 is irradiated from the light source 7 toward the surface portion of the substrate 3, and fluorescence is detected by the photodetector 9. At this time, if the predetermined fluorescence is detected by the photodetector 9, the binding between the secondary antibody 6 and the antigen 2, that is, the presence of the antigen 2 in the sample can be confirmed.

  In the above example, it is the secondary antibody 6 that is actually confirmed by fluorescence detection. However, if the secondary antibody 6 does not bind to the antigen 2, it is washed away and is present on the substrate 3. Since it cannot be obtained, the presence of the antigen 2 as the measurement target substance is indirectly confirmed by confirming the presence of the secondary antibody 6.

  In particular, in recent years, the development of cooled CCDs and other improvements in the performance of photodetectors has made progress, and the fluorescence method described above has become an indispensable means for biological research. Widely used in the field. Particularly in the visible region, for example, FITC (fluorescence wavelength: 525 nm, quantum yield: 0.6) or Cy5 (fluorescence wavelength: 680 nm, quantum yield: 0.3) is a practical guideline of 0.2. Fluorescent dyes with a high quantum yield exceeding the above have been developed, and it is expected that the application field of the fluorescence method will be further expanded.

  Conventionally, a fluorescence method using evanescent light has also been proposed. An example of a fluorescence measuring apparatus that implements this method is schematically shown in FIG. In FIG. 11, the same elements as those in FIG. 10 are denoted by the same reference numerals, and description thereof will be omitted unless necessary (the same applies hereinafter).

  In this fluorescence measuring device, a prism (dielectric block) 13 is used as an alternative to the substrate 3 described above, and the primary antibody 4 is applied to the prism 13. Then, the excitation light 8 from the light source 7 is irradiated through the prism 13 under the condition of total reflection at the interface between the prism 13 and the liquid sample 1. In this configuration, when the excitation light 8 is totally reflected at the interface, the secondary antibody 6 is excited by the evanescent light 11 that leaks out in the vicinity of the interface. Fluorescence detection is performed by a photodetector 9 disposed on the opposite side of the liquid sample 1 from the prism 13 (upward in the drawing).

  In this fluorescence measuring apparatus, the excitation light 8 is incident at an angle that totally reflects from below in the figure, thereby generating evanescent light 11 that reaches only a few hundred nm from the interface to excite the secondary antibody 6. Therefore, the excitation light 8 reflected and scattered by the liquid sample 1, the liquid sample 1 excited by the excitation light 8, and the fluorescence (autofluorescence) emitted from the container reach the photodetector 9 and back against the fluorescence signal to be detected.・ The problem of grounding can be minimized. Therefore, this evanescent fluorescence method has attracted attention as a method that can significantly reduce (light) noise compared to conventional fluorescence methods and can measure the measurement target substance in units of one molecule.

  As one of the apparatuses for carrying out the fluorescence method as described above, as described in Patent Document 1, for example, a light source that emits excitation light, and the excitation light is propagated inside and emitted from the outer surface, and a sample A sensor unit made of rod-like or plate-like glass that excites a phosphor indicating the presence of a substance to be measured in the liquid with excitation light, and a photodetector that detects the fluorescence emitted from the phosphor by the excitation. What is provided is known. When performing measurement with this type of apparatus, the sensor unit is usually immersed in the sample solution, and the phosphor is excited by excitation light emitted from the sensor unit into the sample solution. The fluorescence generated thereby is detected by the photodetector.

  Patent Document 2 and Patent Document 3 show that an optical fiber is applied as the sensor unit as described above. In particular, Patent Document 2 also describes that the phosphor is excited by evanescent light leaking from the surface of the optical fiber.

As described above, the fluorescence measurement device using the sensor unit immersed in the sample liquid is compared with a fluorescence measurement device in which the sensor unit is incorporated in a part of the liquid tank and the sample liquid is introduced therein using a pump or the like. And the configuration is simplified and it can be formed at low cost.
U.S. Pat. No. 4,703,182 Japanese Patent No. 1916924 JP 2006-047250 A

  However, the conventional fluorescence measuring apparatus using the sensor unit immersed in the sample solution is sufficient when it is desired to measure a very small amount of a measurement target substance, for example, about 1 p · mol (pico mol) / l (liter). The problem is that accurate measurement accuracy cannot be obtained. This problem is caused when whole blood, serum, or urine that contains a large amount of absorption / scattering components other than the measurement object is the measurement object, and in the case of an optical fiber with a cladding or coating sensor. However, it is conspicuously recognized in the case of a rod-shaped material made of cheaper glass, integrally molded transparent resin, or the like. This is because the excitation light and the received light are scattered or absorbed and attenuated by contacting with the measurement object at the interface while propagating through the glass or the like serving as an optical waveguide. In order to reduce the influence, generally, an optical fiber with a clad or a coating may be used. However, in the above fields where disposables are required, the consumables become expensive and cannot be adopted from the viewpoint of cost.

  In addition, if a process for cleaning and removing unnecessary components is provided, an inexpensive rod-shaped sensor unit can be adopted, but an expensive liquid delivery mechanism such as a dispenser or pump is required for cleaning, and the device is It becomes expensive.

  In addition, the sensor unit in which fluorescence measurement is performed in a state immersed in a solution such as a sample solution, the reflectivity or optical path of the excitation light / received light at the interface depending on the method of insertion into the solution, the amount of the solution, and the liquid shaking. It is difficult to maintain the reproducibility of measurement.

  The present invention has been made in view of the above circumstances, and uses a sensor unit that is inexpensive and can be consumed as a disposable product, and is colored in a sample such as whole blood, serum, or urine, or a sample that scatters light. It is an object of the present invention to provide an inexpensive fluorescence measuring apparatus that can measure a trace amount of the component and can maintain the reproducibility of the measurement.

The fluorescence measurement apparatus according to the present invention is a type of fluorescence measurement apparatus in which a sensor unit that propagates excitation light is immersed in a sample solution. The excitation light or fluorescence is absorbed by the sample solution or the like attached to the outer surface of the sensor unit, or It is designed to prevent scattering, and specifically,
A light source that emits excitation light;
A sensor unit that causes excitation light incident from one end to propagate inside, emits evanescent light from the other end, and excites a phosphor that indicates the presence of a substance to be measured in a sample solution immersed in the other end by excitation light; ,
In a fluorescence measuring device comprising a photodetector for detecting fluorescence emitted from a phosphor by excitation,
The sensor unit is a substantially columnar sensor unit body;
A cylindrical cover part surrounding the sensor part body via a space between at least the outer peripheral surface adjacent to the other end of the sensor part body,
The cover portion has a closing portion that closes the space at the end portion on the other end side.

  The “space” may be an air layer or a vacuum.

  In the present invention, a reinforcing rib for bridging the portion and the cover portion may be formed in a portion where the excitation light of the sensor portion main body is not reflected.

  Moreover, it is preferable that the cover part is shape | molded or painted with the material which absorbs excitation light and / or fluorescence.

  This cover part is preferably formed by integral molding with the sensor part main body. Moreover, this cover part may be provided with the engaging part engaged with the holding | maintenance part of the holding | maintenance apparatus which hold | maintains a sensor part in a predetermined position.

  Moreover, in this invention, it is preferable that a sensor part main body is what propagates excitation light by a waveguide mode.

  Moreover, it is preferable that a ligand that binds to the measurement target substance is fixed to the outer surface of the other end of the sensor unit.

  In the fluorescence measuring apparatus of the present invention, necessary reagents, sensor units, reaction tanks and the like may be supplied and discarded as a set.

  The present inventor has found that the above-mentioned problem recognized in the conventional fluorescence measuring apparatus, that is, the problem that sufficient measurement accuracy cannot be obtained when an inexpensive sensor unit is used and it is desired to measure a trace amount of a measurement target substance with an inexpensive apparatus. When the sample liquid is present around the sensor part immersed in the sample liquid, the excitation light or fluorescence is absorbed or scattered by the sample liquid, so that it is difficult for the photodetector to detect the excitation light. I found out that it is caused by.

  Based on this new knowledge, the fluorescence measuring apparatus according to the present invention has a sensor unit with a space between a substantially columnar sensor unit body and an outer peripheral surface adjacent to at least the other end of the sensor unit body from which the evanescent light is emitted. And a cylindrical cover portion surrounding the sensor portion main body, and the space is closed at the end portion on the other end side of the cover portion, so that the liquid level is positioned below the upper end of the cover portion. If the sensor part is immersed in the sample liquid, the cover part comes into contact with the sample liquid, and the sensor part body where the excitation light and received light propagate inside is directly on the other end side surface where the evanescent light is emitted, that is, the sensing part directly. Since it comes into contact with the sample solution, it is possible to prevent the outer peripheral surface of the sensor unit body from coming into direct contact with the sample solution.

  Accordingly, the influence of the absorption and scattering is eliminated, and a very small amount of a measurement target substance can be measured with sufficient accuracy. In addition, since the outer peripheral surface of the sensor unit body is not in contact with a solution such as a sample solution, when performing fluorescence measurement while immersed in the solution, the sensor unit is totally reflected by the method of insertion into the solution, the amount of the solution, and the liquid shaking. Therefore, the reproducibility of the measurement can be maintained. Further, since the space can serve as a cladding layer, it is not necessary to use an expensive optical fiber having a cladding layer in the sensor portion. Thereby, a sensor part can be made cheap.

  Further, in the case where a reinforcing rib for bridging the portion and the cover portion is formed in a portion where the excitation light of the sensor portion main body is not reflected, the strength of the cover portion can be reinforced.

  In addition, when the cover part is molded or painted with a material that absorbs excitation light and / or fluorescence, for example, the cover part may scatter light generated by scattering of excitation light or fluorescence due to impurities or the like in the sensor body. Since absorption is possible, detection of scattered light by the photodetector can be prevented.

  Further, when the cover part is formed by integral molding with the sensor part main body, the sensor part can be formed at a low cost, so that it is most suitable for use in a diagnostic device with a high demand for single use. .

  Further, when the cover portion includes an engaging portion that engages with a holding portion of a holding device that holds the sensor portion in a predetermined position, the sensor portion can be easily moved to the predetermined position without changing the condition of total reflection. It becomes possible to arrange | position to.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is a conceptual diagram showing a schematic configuration of a fluorescence measuring apparatus 100 according to an embodiment of the present invention. FIG. 2 is a front sectional view (a) and a side view (b) of a sensor unit 51 of the fluorescence measuring apparatus 100 of FIG. is there. In FIG. 1 and FIG. 2, for the sake of convenience, the side where the light source 50 is located, that is, the upper side of the drawing will be described as the upper side.

  As shown in FIG. 1, the fluorescence measuring apparatus 100 according to this embodiment includes a light source 50 such as a semiconductor laser that emits excitation light 50a having a wavelength of 635 nm, a collimator lens that adjusts the light flux of the excitation light 50a emitted from the light source 50, and the like. The optical system 21, the sensor unit 51 having an optical waveguide for propagating excitation light 50a and fluorescence 59, which will be described later, and a dichroic mirror 22 that transmits, for example, light having a wavelength of 645 nm or less and reflects light having a wavelength of 645 nm or more at a right angle. And a photodetector 37 such as a photodiode for detecting light reflected by the dichroic mirror 22.

  The sensor unit 51 is formed using, for example, a transparent resin having a refractive index n1 = 1.7. Specifically, a phenol resin (phenol-formaldehyde) (“Plastic Vol 52 No. 8” by Takeo Yasuda, p98- It is formed using a test method and an evaluation result <19> of each dynamic characteristic of 101 plastic material, more specifically, “Teslalid” (registered trademark), “Iry” (registered trademark), etc. manufactured by HOYA.

  A characteristic of the present invention is that the sensor unit 51 is formed between a substantially cylindrical sensor unit main body 52 and an outer peripheral surface 52c adjacent to at least the lower end of the sensor unit main body 52, as shown in FIGS. And a cylindrical cover portion 53 that surrounds the sensor portion main body 52 via the space A, and the cover portion 53 has a closing portion 53a that closes the space A at the lower end side end portion. . In this embodiment, the space A is an air layer A, and will be described as an air layer A hereinafter.

  The sensor unit main body 52 is formed of the above-mentioned material into a substantially cylindrical shape with a diameter W = approximately 10 mm, for example, and the upper end side is, for example, θ0 = between the upper end surface 52a when viewed from the front, as shown in FIG. Inclined surfaces 52b having an angle of approximately 53 degrees are formed on both the left and right sides. The sensor unit main body 52 and the cover unit 53 are formed by integral molding with their lower end surfaces being substantially flat, that is, with the lower end surface 51a of the sensor unit 51 being substantially flat. Thus, when the cover part 53 is formed by integral molding with the sensor part main body 52, since the sensor part 51 can be formed at low cost, it is adopted in a diagnostic apparatus that is highly required to be disposable. Ideal for.

  Since the sensor unit 51 has a refractive index n1 = 1.7 as described above, the critical angle θ = 36.03 degrees and the refractive index n3 = 1 if the periphery of the sensor unit 51 is, for example, air with a refractive index n2 = 1. If the physiological saline is .335, the critical angle θ is 51.75 degrees, and the lower end of the sensor unit 51 is positioned below the upper end surface of the cover unit 53 as shown in FIG. When immersed in physiological saline, if the excitation light 50a is incident perpendicularly to the inclined surface 52b, the angle between the horizontal plane and the light beam on the peripheral surface of the sensor unit body 52 is θ1 = 37 degrees, and the lower end surface 51a of the sensor unit 51 The angle θ2 formed by the vertical plane and the light beam is 53 degrees, and the angle θ1 = 37 degrees is larger than the critical angle θ = 36.03 degrees on the peripheral surface of the sensor unit main body 52 where air exists in the surroundings. At the lower end surface 51a where the salt solution exists, the angle θ Since = 53 degrees greater than the critical angle theta = 51.75 °, the excitation light 50a will be totally reflected by the lower end surface 51a of the sensor body 52 inside and the sensor unit 51.

  At this time, for example, when it is desired to excite a phosphor described later on the lower end surface 51a of the sensor unit 51 after four total reflections inside the sensor unit main body 52, as shown in FIG. The height H2 to the lower end of 52b is H2 = 4 × W × tan θ1 = 30.14 mm. Note that the values of the angle θ0, the diameter W, and the height H2 of the sensor unit body 52 are such that excitation light and fluorescence are totally reflected on the outer peripheral surface of the sensor unit body 52 and the lower end surface 51a of the sensor unit 51 during fluorescence measurement. Or the refractive index of the solution or the like in which the sensor unit 51 is immersed.

  As an example, the fluorescence measuring apparatus 100 of the present embodiment is a CRP antigen (molecular weight 110,000 Da), and a primary antibody (monoclonal antibody) that specifically binds to it is above the lower end surface 51a. Fixed to. This primary antibody is fixed to the lower end surface 51a by, for example, an amine coupling method via PEG having a terminal carboxyl group. On the other hand, as the secondary antibody, a monoclonal antibody labeled with a phosphor (Cy5 Ge-healthcare) (epitope; antigenic determinant is different from the primary antibody) is used.

  The amine coupling method includes the following steps (1) to (3) as an example. This is an example when a 30 μl (microliter) cuvette / cell is used.

(1) Activate the -COOH group at the end of the linker (terminal) 30 μl of an equal volume mixed solution of 0.1 M (mol) NHS and 0.4 M EDC was added and left at room temperature for 30 minutes. In addition,
NHS: N-hydrooxysuccinimide
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
It is.

(2) Immobilization of primary antibody
After washing 5 times with PBS buffer (pH 7.4), add 30 μl of the primary antibody solution (500 μg / ml) and let stand for 30-60 minutes at room temperature. (3) Block unreacted -COOH groups
After washing 5 times with PBS buffer (pH 7.4), add 30 μl of 1M ethanolamine (pH 8.5) and let stand at room temperature for 20 minutes. Wash 5 times with PBS buffer (pH 7.4).

  The light source 50 is not limited to the semiconductor laser, and other known light sources such as LEDs can be appropriately selected and used. The photodetector 37 is not limited to the above-described one, and a known one such as a CCD, a photomultiplier tube, or c-MOS can be appropriately selected and used. If the excitation wavelength is changed, a dye other than Cy5 can be used as a label.

  Hereinafter, the operation of the fluorescence measuring apparatus 100 configured as described above will be described in detail with reference to FIG. First, the sensor unit 51 is immersed in plasma as a sample solution. At this time, as shown in FIG. 1, the sample liquid is immersed so that the liquid surface is located below the upper end surface of the cover portion 53. By doing so, the outer peripheral surface of the sensor unit main body 52 is in contact with the atmosphere or the air layer A, and only the lower end surface 51a of the cover unit 53 and the sensor unit 51 is in direct contact with the plasma, so that excitation light and received light propagate inside. It is possible to prevent plasma from being present on the outer peripheral surface of the sensor unit main body 52. If the CRP antigen is contained in the plasma, the CRP antigen and the primary antibody fixed to the lower end surface 51a of the sensor unit 51 are combined. In addition, this coupling | bonding is accelerated | stimulated by moving the sensor part 51 lightly up and down in the state which immersed the sensor part 51. FIG.

  Next, the sensor unit 51 is immersed in the reaction solution containing the secondary antibody labeled with the aforementioned phosphor. At this time, similarly to the above, the reaction solution is dipped so that the liquid level is located below the upper end surface of the cover portion 53, and is lightly moved up and down. By doing so, if the CRP antigen is bound to the primary antibody on the lower end surface 51a of the sensor unit 51, the binding between the CRP antigen and the secondary antibody is promoted.

  After that, the sensor unit 51 is immersed in the buffer solution and moved up and down to clean the lower end surface 51a of the sensor unit 51. As a result, if the CRP antigen is bound to the secondary antibody labeled with the fluorescent substance, the excess except for them is washed away.

  Next, fluorescence measurement is performed with the sensor unit 51 immersed in the buffer solution as shown in FIG. At the time of measurement, the light source 50 is driven, and excitation light 50a such as laser light is emitted therefrom. Most of the excitation light 50a travels downward in the waveguide mode while repeating total reflection inside the sensor section main body 52 at the interface between the surrounding surface and the atmosphere or the air layer A. Thus, the excitation light 50a propagated in the sensor section main body 52 reaches the lower end surface 51a of the sensor section 51 and is totally reflected here. Note that the values of the angle θ0, the diameter W, and the height H2 of the sensor unit main body 52 are the refractive indexes of the buffer liquid so that excitation light and fluorescence are totally reflected on the outer peripheral surface of the sensor unit main body 52 and the lower end surface 51a of the sensor unit 51. It is determined according to n3.

  At this time, evanescent light oozes out at the interface between the lower end surface 51a of the sensor unit 51 and the buffer solution. Therefore, if the CRP antigen is bound to the primary antibody on the lower end surface 51a, the secondary antibody in the reaction solution is further bound to the antigen, and the fluorescent substance that is the label for the secondary antibody is the evanescent light. Excited by The excited phosphor emits fluorescence 59 having a predetermined wavelength, and at least a part of the emitted fluorescence 59 propagates inside the sensor unit main body 52 and is reflected by the dichroic mirror 22 at a right angle and detected by the photodetector 37. Is done.

  Thus, when the light detector 37 detects the fluorescence 59 having a predetermined wavelength, it is confirmed that the secondary antibody is bound to the CRP antigen, that is, that the CRP antigen is contained in the plasma as the sample liquid. It becomes possible to confirm. It is also possible to detect the concentration of the substance to be measured from the intensity of the signal that has detected the fluorescence 59 as described above. The fluorescence measuring apparatus 100 performs the measurement as described above.

  As described above, in the fluorescence measuring apparatus 100 of the present embodiment, the sensor unit 51 includes the substantially columnar sensor unit main body 52 and the cover unit 53, so that the liquid level is positioned below the upper end of the cover unit 53. If the sensor unit 51 is immersed in the sample solution as described above, the cover unit 53 comes into contact with the sample solution, and the sensor unit main body 52 through which excitation light and light reception light propagates is the lower end surface 51a from which evanescent light is emitted, that is, sensing. Since only the portion is in direct contact with the sample liquid, it is possible to prevent the outer peripheral surface of the sensor section main body 52 from being in direct contact with the sample liquid.

  Therefore, the influence of excitation light and fluorescence absorption and scattering by the sample liquid is eliminated, and a very small amount of a measurement target substance can be measured with sufficient accuracy. In addition, since the outer peripheral surface of the sensor unit main body 52 is not in contact with a solution such as a sample solution, when performing fluorescence measurement in a state immersed in the solution, the sensor unit 51 may be inserted into the solution, the amount of the solution, and the liquid fluctuation. Since the conditions for total reflection do not change, measurement reproducibility can be maintained. Further, since the air layer A can serve as a cladding layer, it is not necessary to use an expensive optical fiber having a cladding layer for the sensor unit 51. Thereby, the sensor unit 51 can be made inexpensive.

  In the sensor unit 51 of the fluorescence measuring apparatus 100 of the present embodiment, the space between the outer peripheral surface 52c of the sensor unit main body 52 and the cover unit 53 is the air layer A, but the present invention is not limited to this. For example, when the above measurement is performed in a vacuum, the space may be a vacuum, or the end on the upper end side of the cover portion 53 may be closed to make the space a vacuum, or may be changed as appropriate. Is possible.

  In the fluorescence measuring apparatus 100 of the present embodiment, the dichroic mirror 22 transmits light having a wavelength of 645 nm or less and reflects light having a wavelength of 645 nm or more at a right angle. However, the present invention is not limited to this. For example, light having a wavelength of 645 mm or more may be transmitted, and light having a wavelength of 645 mm or less may be reflected at a right angle. In this case, the positions of the light source 50 and the optical system 21 and the photodetector 37 in FIG. 1 are reversed, and the light transmitted through the dichroic mirror 22 is detected by the photodetector 37.

  Further, in the fluorescence measuring apparatus 100 of the present embodiment, a so-called double-pass method is adopted in which the photodetector 37 is disposed on the upper side as means for measuring fluorescence, and the fluorescence 59 propagated inside the sensor body 52 is detected. However, the fluorescence measuring apparatus according to the present invention is not limited to this, and employs a so-called single-pass method in which, for example, the photodetector 37 is disposed below and the fluorescence 59 emitted from the phosphor is directly detected. May be.

  Moreover, although the fluorescence measuring apparatus 100 of this embodiment shall perform the said fluorescence measurement in the state immersed in the buffer solution, the fluorescence measuring apparatus of this invention is not restricted to this, For example, air, a sample liquid, Even if it measures in the state immersed in solutions, such as a pure water, the effect similar to the above can be acquired. In this case, the sensor unit 51 and the solution are configured to have a condition that the excitation light 50 a is totally reflected by the lower end surface 51 a of the sensor unit 51.

  Next, the fluorescence measuring apparatus 100-2 according to the second embodiment of the present invention will be described in detail with reference to the drawings. FIG. 3 shows the side shape of the fluorescence measuring apparatus 100-2 according to the second embodiment, and FIG. 4 shows the planar shape of the turret 30 used therein. As shown in the figure, the fluorescence measuring apparatus 100-2 includes the turret 30, a pedestal 31 that holds the turret 30 in a horizontal state and is rotatably supported, a vertical member 32 that stands vertically from the pedestal 31, and It has a vertical moving table 33 that is held by a vertical member 32 and is movable in the vertical direction by a driving means (not shown). The gantry 31, the vertical member 32, and the vertical moving table 33 are collectively referred to as a holding device.

  A stepping motor 34 is fixed to the gantry 31, and a turret 30 is fixed to the drive shaft 35. Therefore, when the stepping motor 34 is driven, the turret 30 rotates around the drive shaft 35, that is, in a horizontal plane. Further, a photodetector 37 such as a photodiode is attached to the upper surface of the gantry 31 via a fixture 36. As an example, a pair of photodetectors 37 that are opposed to each other with a space therebetween is used.

  As clearly shown in FIG. 4, a sample tank 40, a reaction tank 41, a buffer liquid tank 42, and a sensor unit holding hole 43 are formed on the upper surface of the turret 30. Further, a through hole 44 is formed in the turret 30. The tanks 40 to 42, the sensor part holding hole 43, and the through hole 44 are arranged at predetermined angular intervals with each center positioned on one common circle around the rotation axis of the turret 30. ing. On the vertical movement table 33, for example, a light source 50 such as a semiconductor laser that emits excitation light 50a having a wavelength of 635 nm, and a chuck (holding unit) 49 that releasably holds the sensor unit 51-2 are mounted.

  FIG. 5 is a front sectional view of the sensor unit 51-2 of the present embodiment. Since the sensor unit 51-2 of the present embodiment has substantially the same configuration as the sensor unit 51 of the above-described embodiment, the same portions are denoted by the same reference numerals, description thereof is omitted, and only different portions will be described.

  The sensor unit 51-2 of the present embodiment is different from the sensor unit 51 of the above embodiment in the shape of the cover unit 53. As shown in FIG. 5, the cover portion 53-2 of the present embodiment has an engagement portion 53 b that engages with the chuck portion 49 that projects outwardly in a pin shape on the outer peripheral surface on the upper end side. It is provided with a plurality. By providing the engaging portion 53b, the chuck portion 49, that is, the above-described holding device can be easily attached and detached without considering the optical path and the like, and the sensor portion 51-2 is easily disposed at a predetermined position. Is possible. In addition, the cover part 53-2 of this embodiment is provided with the engaging part 53b of the said shape, However, The cover part of this invention is not restricted to this, If it is the structure which does not affect an optical path as much as possible, It can be appropriately changed according to the shape of the chuck portion 49 of the holding device. The cover portion 53-2 is a hole portion of the turret 30 such that the liquid surface of the sample solution is positioned below the upper end surface of the cover portion 53-2 when the sensor portion 51-2 is inserted into the sample solution. It is formed in consideration of the depth and the like.

  The driving of the stepping motor 34, that is, the rotating operation of the turret 30 is controlled in synchronism with the driving of the vertical moving table 33, the light source 50 and the photodetector 37 by a control circuit (not shown). The chuck 49 includes a mechanical gripping mechanism, which will be described later, as an example, and has a cylindrical lower end portion. The inside of the chuck 49 communicates with air discharge means such as a blower (not shown). Therefore, the holding mechanism 53b is held by the gripping mechanism, and a function of stirring the sample liquid or the like by discharging air is provided. These functions will be described in detail later.

  Note that the fluorescence measurement apparatus 100-2 of the present embodiment is the measurement target because it is the same as that of the above-described embodiment, and thus the description thereof will be omitted. Hereinafter, with respect to the operation of the fluorescence measurement apparatus 100-2, the steps during measurement are described. This will be described with reference to FIG. In FIG. 6, in order to avoid complication of the figure, among the elements shown in FIG. 3, only the minimum elements necessary for explanation in each of the steps (1) to (11) are numbered. Is given.

  First, in this measurement, the sensor unit 51-2 is stored in the sensor unit holding hole 43 in advance. As shown in FIG. 1A, a predetermined amount of whole blood 56 as a sample solution is injected into the sample tank 40, and the turret 30 is rotated by a predetermined angle, so that the sample tank 40 is brought into a position directly below the chuck 49. Is set. Next, as shown in (2), the chuck 49 (that is, the vertical moving table 33) is lowered, and the whole blood 56 is filtered by the filter 48 in the sample tank 40 by the air pressure between the chuck 49 and the sample tank 40. Then, the whole blood 56 is separated into heavy blood cells 57 and plasma 58 as shown in (3).

  After the chuck 49 is pulled up in this state, the turret 30 is further rotated by a predetermined angle, and as shown in (4), the sensor unit 51-2 housed in the sensor unit holding hole 43 is attached to the chuck 49. Set directly below. In this state, the chuck 49 is moved downward, and the sensor portion 51-2 is held by the chuck 49 by sandwiching the engaging portion 53b with a general drilling machine sandwiching the drill. Then, as shown in (5), the chuck 49 is pulled up, and the sensor unit 51-2 is pulled out from the sensor unit holding hole 43.

  When the sensor unit 51-2 is completely separated from the turret 30 as shown in (6), the turret 30 is rotated by a predetermined angle, and as shown in (7), the sample tank 40 is again set to a position directly below the chuck 49. The In this state, the chuck 49 is moved down by a predetermined length and is moved up and down, so that the sensor unit 51-2 held therein is lightly moved up and down while being immersed in the plasma 58. At this time, similarly to the above embodiment, the plasma 58 is immersed and moved up and down so that the liquid level of the plasma 58 is located below the upper end surface of the cover portion 53-2, and the plasma adheres to the outer peripheral surface of the sensor portion main body 52. To prevent. By moving up and down in this way, if CRP antigen is contained in the plasma 58, the binding between the CRP antigen and the primary antibody fixed to the lower end surface 51a of the sensor unit 51-2 is achieved. Promoted.

  Thereafter, the sensor unit 51-2 is pulled up from the sample tank 40, and then the turret 30 is rotated by a predetermined angle, whereby the reaction tank 41 is set to a position directly below the chuck 49 as shown in (8). In the reaction tank 41, a reaction solution 45 containing the secondary antibody labeled with the above-described phosphor is stored. Next, the chuck 49 is moved down by a predetermined length and thereby moved up and down lightly while the sensor unit 51-2 is immersed in the reaction solution 45.

  At this time, in the same manner as described above, the reaction liquid 45 is immersed and moved up and down so that the liquid surface of the reaction liquid 45 is located below the upper end surface of the cover section 53-2, and the reaction liquid 45 adheres to the outer peripheral surface of the sensor section main body 52. To prevent. By moving up and down in this way, if the CRP antigen is bound to the primary antibody on the lower end surface 51a of the sensor unit 51-2, the binding between the CRP antigen and the secondary antibody is promoted.

  Thereafter, the sensor unit 51-2 is pulled upward from the reaction tank 41, and then the turret 30 is rotated by a predetermined angle, whereby the buffer liquid tank 42 is set to a position directly below the chuck 49 as shown in (9). . Next, the chuck 49 is moved down by a predetermined length and then moved up and down, whereby the sensor unit 51-2 is washed with the buffer liquid 46 stored in the buffer liquid tank. In other words, if the CRP antigen is bound to the secondary antibody labeled with a fluorescent substance, the excess except for them is washed away.

  Thereafter, the sensor unit 51-2 is pulled up from the buffer liquid tank 42, and then the turret 30 is rotated by a predetermined angle, whereby the through hole 44 is set to a position directly below the chuck 49 as shown in (10). . Next, the chuck 49 is lowered, so that the sensor unit 51-2 is positioned in the through hole 44. In this state, since only air exists around the sensor unit 51-2, the sensor unit 51-2 substantially absorbs excitation light and fluorescence when performing fluorescence measurement described below. It will be arranged in an atmosphere that does not scatter.

  The fluorescence measurement performed in this state will be described in detail with reference to FIG. 3 showing the state at that time. At the time of measurement, the light source 50 is driven, and excitation light 50a such as laser light is emitted therefrom. Most of the excitation light 50a travels downward in the waveguide mode while repeating total reflection in the sensor section main body 52 at the interface between the outer peripheral surface and air. A part of the excitation light 50a propagated in the sensor unit main body 52 thus reaches the lower end surface 51a of the sensor unit 51-2 and is totally reflected there.

  At this time, evanescent light oozes out from the interface between the lower end surface 51a of the sensor unit 51 and the air. Therefore, if the CRP antigen is bound to the primary antibody on the lower end surface 51a, the secondary antibody in the reaction solution 45 is further bound to the antigen, and the phosphor that is the label for the secondary antibody becomes the above evanescent. Excited by light. The excited phosphor emits fluorescence 59 having a predetermined wavelength, and the fluorescence 59 is detected by the photodetector 37. Thus, when the photodetector 37 detects the fluorescence 59 having a predetermined wavelength, the secondary antibody is bound to the CRP antigen, that is, the CRP antigen is contained in the plasma 58 as the sample liquid. Can be confirmed. It is also possible to detect the concentration of the substance to be measured from the intensity of the signal that has detected the fluorescence 59 as described above.

  When the fluorescence measurement is thus completed, the sensor unit 51-2 is pulled upward from the through hole 44, and then the turret 30 is rotated by a predetermined angle, so that the sensor unit holding hole is shown in FIG. 43 is set at a position directly below the chuck 49. Next, the chuck 49 is lowered, whereby the sensor unit 51-2 is inserted into the sensor unit holding hole 43. Next, the chuck 49 is opened, the chuck 49 is moved up, and the sensor unit 51-2 is detached therefrom. Thus, the sensor unit 51-2 returns into the holding hole 43. Thereafter, the used turret 30 is discarded and replaced with a new turret 30 so that the measurement can be performed without worrying about contamination.

  Here, in the present embodiment, as is apparent from the above description, the sensor unit driving unit is configured by the turret 30 as the rotating unit and the reciprocating unit including the vertical movement table 33 and the chuck 49. When such a turret 30 is used, measurement can be performed very efficiently. However, the sensor unit driving means is not limited to this, and can be appropriately configured using other known mechanisms.

  If there is no need to worry about contamination, the sensor unit 51-2 and the like can be washed and stored again to re-use the reagent.

  Note that when the fluorescence measurement is performed, the plasma 58 and the reaction liquid 45 do not adhere to the outer peripheral surface of the sensor unit main body 52 as described above. Therefore, the excitation light 50a and the fluorescence 59 are absorbed and scattered by the plasma 58 and the reaction liquid 45. Therefore, a trace amount of a substance to be measured can be measured with sufficient accuracy. Furthermore, since the sensor unit 51-2 is disposed in an atmosphere that does not substantially absorb and scatter the excitation light 50a and the fluorescence 59, the fluorescence 59 is absorbed or scattered. It can prevent that the intensity | strength of the fluorescence detected falls and the measurement precision of a measuring object substance is impaired. In addition, it is possible to prevent the excitation light 50a partially leaking from the sensor unit 51-2 from being scattered and entering the photodetector 37, thereby impairing measurement accuracy.

  In addition, although the fluorescence measuring apparatus 100-2 of the present embodiment includes the turret 30 as described above, the present invention is not limited to this, and may include, for example, a plurality of containers or may be changed as appropriate. Is possible.

  In the present embodiment, the atmosphere that does not substantially absorb and scatter the excitation light 50a and the fluorescence 59 is air, and the phosphor is excited and detected in the atmosphere. Liquids such as pure water and PBS buffer are also suitable as such an atmosphere, and the fluorescence measuring apparatus of the present invention may be configured to perform excitation and fluorescence detection of a phosphor in such a liquid. .

  The fluorescence measuring apparatuses 100 and 100-2 according to the embodiments described above detect the secondary antibody by performing fluorescence measurement, thereby indirectly detecting the measurement target substance in the sample solution. Of course, the fluorescence measuring apparatus of the invention can be configured to directly detect a substance to be measured which is a phosphor.

  The sensor unit constituting the fluorescence measuring apparatus of the present invention is not limited to the sensor units 51 and 51-2 described above, and can be appropriately changed. 7 is a front sectional view (a) and a side view (b) of the sensor unit 51-3 of the third embodiment, and FIG. 8 is a front sectional view of the sensor unit 51-4 of the fourth embodiment. 9 shows a front sectional view (a) and a top view (b) of the sensor unit 51-5 of the fifth embodiment, and each will be described below.

  The sensor unit 51-3 of the third embodiment has a configuration substantially similar to that of the sensor unit 51 of the first embodiment, but the shape of the cover unit 53 is different. As shown in FIGS. 7A and 7B, the cover portion 53-3 of the present embodiment is formed in a cylindrical shape whose diameter increases from the lower end toward the upper end. By doing so, it can be easily removed from the mold at the time of integral molding.

  The sensor unit 51-4 of the fourth embodiment has the same shape as the sensor unit 51-2 of the second embodiment. However, as shown in FIG. 8, the cover unit 53-4 includes the excitation light 50a and / or The difference is that it is formed of a material that absorbs fluorescence 59. Specifically, this material is, for example, a molding material used for plastic filters PIR730 and PIR800 manufactured by Annaka Special Glass Mfg. Co., azo dyes commonly used for PC (polycarbonate) and PES (polyethersulfone) A mixture of various dyes can be used. In the sensor unit 51-4, the sensor unit main body 52 and the cover unit 53-4 are integrally formed by two-color molding. In this way, by forming the cover portion 53-4 with a material that absorbs the excitation light 50 a and / or the fluorescence 59, the cover portion 53-4 is excited by, for example, impurities in the sensor portion main body 52 and the excitation light 50 a and fluorescence 59. Therefore, it is possible to prevent the scattered light from being detected by the light detector 37. In addition, although the cover part 53-4 of this embodiment shall be formed with the said material, this invention is not restricted to this, As long as it can absorb the excitation light 50a and the fluorescence 59, it may be painted. .

  The sensor unit 51-5 of the fifth embodiment has a configuration substantially similar to that of the sensor unit 51-2 of the second embodiment. However, as shown in FIGS. The difference is that a reinforcing rib 55 for bridging the portion and the cover portion 53 is provided in a portion where the excitation light 50a of the light is not totally reflected. By providing the rib 55 in this manner, the strength of the cover portion 53 can be reinforced. In the present embodiment, one rib 55 is provided. However, the present invention is not limited to this, and for example, a plurality of ribs 55 may be provided at predetermined intervals in the circumferential direction, or a plurality may be provided in the vertical direction. The excitation light 50a may be provided anywhere as long as it is not totally reflected.

  The height H of the total reflection position can be obtained from the internal reflection angle θ1 in FIG. 1 and the diameter W of the sensor unit main body 52 by the equation H = n × (W / 2) tan θ1. Here, n is an odd integer, and the excitation light 50a is totally reflected at this height position. Therefore, the “non-total reflection position” is a position having a height different from the height of the total reflection position obtained by the above formula. For example, the total reflection position is W = 10 mm, θ1 = 37 degrees, n = In the case of 7, the height H = 7 × (10/2) tan 37 ° = 26.3 mm. Similarly, in the case of n = 5, since the height H = 18.8 mm, the height H = 18.8. Between 26.3 mm is the height of the position where total reflection does not occur. Therefore, for example, the rib 50 can be formed at the position of the height H = 22.6 mm which is the intermediate value.

  Although the fluorescence measurement apparatus according to the embodiment of the present invention has been described above, the fluorescence measurement apparatus of the present invention is not limited to this, and can be appropriately changed without departing from the spirit of the present invention.

The conceptual diagram which shows schematic structure which shows the fluorescence measuring apparatus by one Embodiment of this invention. Front sectional view and side view of the sensor unit of the fluorescence measuring apparatus of FIG. Schematic side view showing a fluorescence measuring apparatus according to a second embodiment of the present invention. FIG. 3 is a plan view showing a part of the fluorescence measuring apparatus of FIG. Front sectional view of the sensor unit of the fluorescence measuring apparatus of FIG. Schematic diagram showing the measurement process by the fluorescence measurement device of FIG. Front sectional view and side view showing a third embodiment of the sensor unit Front sectional view showing a fourth embodiment of the sensor unit Front sectional view and top view showing a fifth embodiment of the sensor unit Schematic side view showing an example of a conventional fluorescence measuring apparatus Schematic side view showing another example of a conventional fluorescence measuring apparatus

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 2 Antigen 4 Primary antibody 6 Secondary antibody 7, 50 Light source 8, 50a Excitation light 9, 37 Photo detector 10 Phosphor 21 Optical system 22 Dichroic mirror 30 Turret 33 Vertical moving table 34 Stepping motor 40 Sample tank 41 Reaction Tank 42 Buffer liquid tank 43 Sensor part holding hole 44 Through hole 49 Chuck part (holding part)
51 Sensor part 52 Sensor part body 53 Cover part 53a Blocking part 59 Fluorescence

Claims (7)

A light source that emits excitation light;
The excitation light incident from one end is propagated by total reflection on the peripheral surface , and evanescent light is emitted from the other end. A sensor unit excited by
In a fluorescence measuring device comprising a photodetector for detecting fluorescence emitted from the phosphor by the excitation,
The sensor unit is a substantially columnar sensor unit body;
A cylindrical cover part surrounding the sensor part body with a space between at least the outer peripheral surface adjacent to the other end of the sensor part body,
The fluorescence measuring apparatus characterized in that the cover portion has a closing portion that closes the space at the end on the other end side. The fluorescence measuring apparatus according to claim 1, wherein a reinforcing rib for bridging the part and the cover part is formed in a part of the sensor part main body where the excitation light is not reflected. The fluorescence measuring apparatus according to claim 1, wherein the cover part is molded or painted with a material that absorbs the excitation light and / or the fluorescence. The fluorescence measuring apparatus according to claim 1 or 3, wherein the cover part is formed by integral molding with the sensor part main body. The fluorescence measurement according to any one of claims 1 to 4, wherein the cover portion includes an engaging portion that engages with a holding portion of a holding device that holds the sensor portion in a predetermined position. apparatus. The fluorescence measuring apparatus according to claim 1, wherein the sensor unit main body propagates excitation light in a waveguide mode. The fluorescence measuring apparatus according to any one of claims 1 to 6, wherein a ligand that specifically binds to the measurement target substance is fixed to an outer surface of the other end of the sensor unit.

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