JP5619690B2 - Fluorescence measuring apparatus and well manufacturing method - Google Patents

Description

  The present invention relates to a fluorescence measurement PCR (Polymerase Chain Reaction) apparatus and the like.

The PCR method is known as a method for growing a target DNA region from a very small amount of DNA in a short time. The principle uses two known sequences on double-stranded DNA to design two primer DNAs that face each other in the direction of synthesis.
When the DNA of the region sandwiched between these primers is synthesized, it becomes a template in the next cycle. By repeating such a reaction, the DNA strand can be amplified geometrically. This method, discovered in 1985, has exploded in popularity due to the use of thermostable enzymes.

  FIG. 11 is a conceptual diagram showing a mode of DNA amplification by the PCR method. First, a template DNA having a DNA region to be amplified as shown in FIG. 11A is composed of two primers and, for example, a heat-resistant DNA polymerase, dNTP (a mixture of dATP, dCTP, dGTP, and dTTP) together with a buffer solution. Placed in the reaction solution.

  In this state, for example, by holding at 95 ° C. for about 2 minutes, the template DNA is separated into single strands by thermal denaturation as shown in FIG.

  Thereafter, the temperature is lowered and maintained at 50 ° C. for 1 minute, for example, so that the primers 101a and 101b are bound (hybridized) to the DNA strand as shown in FIG. 11 (c).

  Next, by holding for 1 minute at the optimal temperature of the enzymatic reaction of DNA polymerase (for example, 72 ° C.), a complementary DNA strand by DNA polymerase is synthesized in the DNA region sandwiched between primers 101a and 101b. As described above, the DNA region sandwiched between the primers 101a and 101b can be amplified twice.

  By repeating the above cycle, only a specific region sandwiched between two primers can be amplified in a large amount among the DNA molecules originally present in the reaction solution.

  This is called a real-time PCR method capable of detecting a PCR reaction during such a reaction. In addition, real-time PCR methods using fluorescent labels include an interlation method and a hybridization method. The hybridization method is less likely to establish nonspecific amplification than the intercalation method, and the target gene chain It is known that the amplification efficiency of the is increased. In addition to the above, the following hairpin primer PCR method and the like are known.

  FIG. 12 is a conceptual diagram of the hairpin primer PCR method. In this method, as shown in FIG. 12A, a hairpin primer 103 in which a DNA sequence forming a hairpin structure is added to the end of the primer is used. A cytosine bulge region to which a small molecule specifically binds is introduced into the hairpin region. Prior to PCR, a low molecular weight compound that emits fluorescence when bound to cytosine bulge is bound.

  As shown in FIG. 12B, when the synthesis of the DNA strand by the polymerase 107 proceeds and the hairpin structure of the hairpin primer 103 is opened by PCR, the cytosine bulge structure disappears. For this reason, the fluorescent dye 105 is liberated. Therefore, the fluorescence intensity decreases. That is, the progress of PCR can be grasped by detecting the fluorescence of the reaction system.

Regarding real-time PCR, for example, a gene amplification method that can detect a gene in a shorter time by using a DNA polymerase having excellent extensibility in PCR has been disclosed (Patent Document 1).

JP 2010-239880 A

  FIG. 13 is a schematic view showing the vicinity of a well for measuring fluorescence by such real-time PCR. First, as shown in FIG. 13A, a reaction solution 113 including the various components described above is placed in a well 109. In this state, light is irradiated from above by the irradiation / light receiving unit 111 (in the direction of arrow I in the figure). The irradiation / light receiving unit 111 is, for example, an optical fiber, and is connected to a light source and a fluorescence measuring device (not shown).

  From the reaction solution irradiated with light, fluorescence corresponding to the progress of PCR is generated (disappears). FIG. 13B is a schematic diagram showing a state in which fluorescence is generated, assuming that the fluorescence generation center 115 is a center where fluorescence is generated. The fluorescence generated from the fluorescence generation center 115 is generated substantially uniformly in all directions (with respect to the spherical direction assuming a sphere centered on the fluorescence generation center 115) around the fluorescence generation center 115 (arrows in the figure). J direction).

At this time, for example, when the distance between the light receiving surface of the irradiation / light receiving unit 111 and the fluorescence generation center 115 is L, the virtual light receiving surface 117 (the surface area of a sphere having a radius L) is represented by 4πL ² . That is, if the reflection at the well 109 is ignored, the fluorescence generated from the reaction solution reaches the entire surface of the virtual light receiving surface 117 having a surface area of 4πL ² uniformly.

Here, if the area of the light-receiving surface of the irradiation / light-receiving unit 111 is a, the amount of fluorescence that can be received among the actually generated fluorescence is the light-receivable region 119 of area a out of the total area of 4πL ^2. (Hatched portion in the figure) only. That is, the fluorescence that has traveled in other directions is dissipated without being received.

Here, the diameter of the irradiation / light receiving unit 111 is 5 mmΦ, and the distance L is 10 mm. Then, the total surface area (4 × π × 10 ² ) of the virtual light receiving surface 117 is about 1257 mm ² . Further, the area of the light receiving surface (π × 2.5 ² ) is about 20 mm ² . Therefore, 20/1257 = about 1.6% with respect to the generated fluorescence.

  Thus, in the conventional fluorescence measuring apparatus, most of the fluorescence actually generated leaks to the outside, and only a small amount of the generated fluorescence can be received. That is, the efficiency of receiving fluorescence is extremely poor. Therefore, for example, in the medical field, it is difficult to ensure detection accuracy unless the amount of antigen (for example, virus) in the sample to be examined is sufficient.

  The present invention has been made in view of such problems. For example, a fluorescence measuring apparatus capable of realizing a sensitivity equivalent to the sensitivity of a virus infection test (15 IU / μL) realized by the Taq Man PCR method, etc. The purpose is to provide.

  In order to achieve the above-mentioned object, the first invention is a fluorescence measurement PCR apparatus, a well for holding a reaction solution, a heater arranged on the outer periphery of the well and capable of adjusting the temperature of the reaction solution, A light source for irradiating light to the reaction solution in the well, a waveguide connected to the lower side of the well, and a fluorescence measuring instrument for measuring fluorescence generated in the well, wherein the well is at least external The resin layer and the inner resin layer have a two-layer structure, and the refractive index of the inner resin layer is larger than the refractive index of the outer resin layer, and the waveguide is made of the same material as the inner resin layer inside. A core portion and a cladding portion made of the same material as the outer resin layer on the outside, the outer resin layer is removed at the lower portion of the well, and the core portion is bonded to the exposed inner resin layer, By the heater By adjusting the temperature, the DNA can be separated, extracted and amplified in the reaction solution, and the fluorescence generated in the well is enclosed in the internal resin layer and led to the core part, and further the optical filter It is possible to measure with the fluorescence measuring instrument via a fluorescent measuring device.

  A plurality of the wells are arranged, the waveguide is connected to an optical switch, and the fluorescence from each of the wells is switched by the optical switch, so that the fluorescence of the plurality of wells can be measured by the fluorescence measuring instrument. Is desirable.

  A reflective film may be formed on the upper end surface of the well. The resin constituting the well is, for example, a heat-resistant polycarbonate resin, modified polycarbonate resin, polyamide, an acrylic polymer with a high Tg component monomer, a norbornene resin, a silicone resin, or a crosslinked acrylic resin. In addition, a fluorine resin or polyamide can be generally used for the outer resin layer. In addition, when the use temperature conditions for heat resistance are not strict, general-purpose thermoplastic resins such as polyethylene, polypropylene, and polystyrene may be used for the internal resin layer, but for PCR, the above heat-resistant resin should be used. Is desirable.

  According to the first invention, since the temperature-controllable heater is provided around the well, DNA separation, extraction, amplification and fluorescence measurement can be performed continuously. Here, the heater to be used may be a heater having an independent shape for each well, or a heater block that heats the entire block containing the well almost uniformly.

  In addition, the well has a two-layer structure, and by making the refractive index of the internal resin layer larger than the refractive index of the external resin layer, the fluorescence from the reaction solution placed in the well is transferred to the internal resin layer of the well. Can be contained. Further, since the internal resin layer is connected to the waveguide at the lower part of the well, fluorescence can be introduced into the waveguide. Therefore, it is possible to efficiently measure fluorescence with a fluorescence measuring instrument connected to the waveguide.

  In addition, by switching the fluorescence generated in a plurality of wells with an optical switch, it is not necessary to install an expensive optical filter and fluorescence measuring instrument for each well.

  Further, by forming a reflective film on the upper end surface of the well, the fluorescence in the inner resin layer does not leak from the upper end surface of the well. In addition, as the resin constituting the well, as the core material resin, polycarbonate (heat resistant temperature 125-135 ° C.), modified polycarbonate (heat resistant temperature 145 ° C.), acrylic polymer with high Tg component monomer, norbornene resin ( By selecting from a heat resistant temperature of 150 ° C., a silicone resin (150-170 ° C.), and a cross-linked acrylic resin (heat resistant temperature of 175 ° C.), wells are not damaged by heat in PCR. Even if PMMA is used as the core material, heat resistance can be improved by using a plastic optical fiber using an α-fluoroacrylate resin as the cladding material.

  When the apparatus is used for a test that does not require heating, a general-purpose plastic fiber using the most commonly used PMMA, polystyrene, or the like can be used as the core material.

  Any resin can be used for the core material as long as it has a lower refractive index than each resin material, is excellent in compatibility or affinity with the core material, and is excellent in heat resistance.

  A second invention is a method for producing a well used in a fluorescence measurement PCR apparatus, wherein the well body has a two-layer structure of an outer resin layer and an inner resin layer having a refractive index larger than that of the outer resin layer. The outer resin layer at the bottom of the well body is removed, the inner resin layer is exposed, the core portion made of the same material as the inner resin layer, and the outer resin on the outside A well manufacturing method comprising: using a waveguide having a clad portion made of the same material as the layer, joining the core portion to the exposed internal resin layer, and joining the clad portion to the external resin layer It is.

  According to the second invention, a well capable of highly sensitive measurement can be easily manufactured.

  According to the present invention, for example, it is possible to provide a fluorescence measuring apparatus capable of realizing a sensitivity equivalent to the sensitivity of a virus infection test (15 IU / μL) realized by the Taq Man PCR method.

Schematic which shows the fluorescence measuring apparatus 1. FIG. (A) is an enlarged view of the well 3, and (b) is a refractive index distribution diagram. It is a cross-sectional enlarged view of a well, (a) is the A section enlarged view of FIG. 1, (b) is the B section enlarged view of FIG. The conceptual diagram which shows the area | region which can receive light with a well. The figure which shows the manufacturing method of a well. The conceptual diagram which shows the temperature cycle of PCR measurement. It is a figure which shows other embodiment, and is the schematic which shows the fluorescence measuring device 1a. The figure which shows the optical switch 47. FIG. (A) is the EE sectional view taken on the line of FIG. 8, (b) is the FF sectional view taken on the line of FIG. The enlarged view of the connection part of the waveguide 9a and the fiber 57. FIG. The conceptual diagram which shows the process of PCR. The conceptual diagram which shows the process of PCR using a hairpin primer. The conceptual diagram which shows the conventional light reception area | region.

  Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic view showing a fluorescence measuring apparatus 1. The fluorescence measuring apparatus 1 mainly includes a plurality of wells 3a, 3b, ..., 3n, a plurality of light sources 5a, 5b, ..., 5n, waveguides 9a, 9b, ..., 9n, and optical. , 11n, fluorescence measuring devices 13a, 13b,..., 13n and a heater 7 for adjusting the temperature of each well. For simplicity, only three wells are shown in the figure. For example, 96 wells are arranged on one plate.

  Wells 3a, 3b,..., 3n (hereinafter collectively referred to as well 3) can hold reaction solution 15 therein. In the well 3, the height of the site for holding the reaction solution is about 10 mm, and the outer diameter is about 8 to 10φ. In addition, waveguides 9a, 9b,..., 9n (hereinafter collectively referred to as waveguide 9) of about 3 to 4 mm are joined to the lower portion of the well 3, respectively.

  Above the well 3, light sources 5a, 5b,..., 5n (hereinafter collectively referred to as the light source 5) are arranged. The light source 5 irradiates the reaction solution in each well 3 with light having a predetermined wavelength. As the light source 5, a laser, LED, or the like can be used.

  FIG. 2A is an enlarged view of the well 3, and FIG. 2B is a distribution diagram of the refractive index including the well. Moreover, Fig.3 (a) is the A section enlarged view of FIG. The well 3 has a two-layer structure of an inner resin layer 19 and an outer resin layer 23 from the side in contact with the reaction solution. Here, as shown in FIG. 2B, the refractive index of the internal resin layer 19 is higher than that of the external resin layer 23.

  In FIG. 2B, L is the refractive index of the region of the reaction solution 15, M is the refractive index of the internal resin layer 19, and N is the refractive index of the external resin layer 23. Note that both sides of N are air layers, and the refractive index is 1.0. The refractive index of the reaction solution 15 depends on the solution used, but is, for example, about 1.4 to 1.55. Moreover, the refractive index of the internal resin layer 19 is about 1.50 to 1.70, for example, and the refractive index of the external resin layer 23 is about 1.48 or less, for example. That is, it is possible to contain light inside the internal resin layer 19.

  The well 3 has, for example, an internal resin layer such as a heat-resistant polycarbonate resin, a modified polycarbonate resin, polyamide, an acrylic polymer with a high Tg component monomer, a norbornene resin, a silicone resin, a crosslinked acrylic resin, etc. It is made of a material selected in consideration of chemical resistance against a reaction solution or the like from a resin having heat resistance of 110 ° C. or higher (more preferably 120 ° C. or higher). Generally, fluororesin or polyamide is used for the external resin layer. Further, the refractive index may be adjusted by changing the resin material constituting each layer, or an additive may be appropriately added to the base material of the same material.

  FIG. 3B is an enlarged view of a portion B in FIG. The waveguide 9 is provided with a core portion 25 at the center and a clad portion 27 at the outer peripheral portion so as to cover the core portion 25. The refractive index of the resin that constitutes the core portion 25 is higher than the refractive index of the resin that constitutes the cladding portion 27. For example, the core portion 25 is made of the same material as the inner resin layer 19, and the clad portion 27 is made of the same material as the outer resin layer 23. A plastic fiber may be used as the waveguide 9.

  At the joint between the well 3 and the waveguide 9, the internal resin layer 19 and the core portion 25 are joined. Further, the outer resin layer 23 and the clad portion 27 are joined. Therefore, the light confined in the internal resin layer 19 is led out to the core portion 25 and propagates in the core portion 25.

  A reflective film 17 is provided on the upper end surface of the well 3. The reflective film 17 prevents light in the well 3 (internal resin layer 19) from leaking from the upper end of the well 3. The reflective film 17 may be formed by vapor deposition of a material such as silicon oxide, titanium oxide, or niobium oxide. Alternatively, a reflective film component may be applied. The method for forming the reflective film 17 is not limited.

  A heater 7 is provided on the outer periphery of the well 3. The heater 7 is connected to a control unit (not shown), and the temperature of the reaction solution in the well 3 can be adjusted. As the heater 7, a heater block may be disposed, or a heater such as resistance heating may be used.

  Optical waveguides 11a, 11b,..., 11n (hereinafter collectively referred to as the optical filter 11) are connected to the waveguide 9, and the fluorescence measuring instruments 13a, 13b,. .. 13n (hereinafter collectively referred to as a fluorescence measuring device 13). The optical filter 11 removes light (for example, irradiation light component) other than the fluorescent component generated in the reaction solution 15. The fluorescence that has passed through the optical filter 11 is measured by each fluorescence measuring device 13.

  That is, in the reaction solution 15 inside the well 3, the above-described DNA amplification is performed by adjusting the temperature by the heater 7. The fluorescent component observed at this time is enclosed in the internal resin layer 19 inside the well 3. The fluorescence in the internal resin layer 19 is led to the waveguide 9 and measured by the optical measuring device 13 through the optical filter 11. As described above, increase / decrease in fluorescence can be monitored in real time.

  FIG. 4 is a diagram showing the diffusion state of the fluorescence generated from the reaction solution when the well 3 is used, as in FIG. Here, the distance from the light source 5 to the fluorescence generation center 29 is L. When the reaction solution is irradiated with light from the light source 5, the fluorescence generated from the reaction solution diffuses substantially uniformly from the fluorescence generation center 29 in all directions (arrow C in the figure).

  The fluorescence generated from the fluorescence generation center 29 reaches the entire surface of the virtual light receiving surface 31 which is a spherical surface having a radius L substantially uniformly. However, when the light source 5 also serves as a light receiving part (in the case of conventional measurement as shown in FIG. 13), the fluorescence that can be received is only the part corresponding to the area of the light receiving part as described above.

  On the other hand, in the present invention, the fluorescence is confined in the well 3 (inner resin layer 19) except for the fluorescence diffused upward from the inner surface of the well 3. Further, as described above, since the reflective film 17 is formed in the upper surface direction of the well 3, all the light confined in the well 3 is emitted to the waveguide 9 below the well 3.

  That is, by using the well 3 of the present invention, all light other than light leaking from the opening of the well 3 can be contained in the well 3 on the virtual light receiving surface 31. For this reason, in the virtual light receiving surface 31, the light receivable region 33 is in all directions (regions indicated by hatching in the drawing) other than the opening of the well 3. Therefore, it is possible to measure the generated fluorescence extremely efficiently compared to the conventional case.

  Next, a method for manufacturing the well 3 will be described. FIG. 5 is a diagram showing a manufacturing process of the well 3. First, as shown in FIG. 5A, the well body 35 is formed. As described above, the well body 3 has a two-layer structure of the inner resin layer 19 and the outer resin layer 23. For example, the well body 3 is molded by injection molding, press molding, or resin injection into a mold. The method may be appropriately selected depending on the type of resin. Moreover, the external resin layer may be configured in two layers.

  Next, as shown in FIG. 5B, a part of the lower surface of the well body 35 is cut to form an external resin layer removal portion 37. In the outer resin layer removing portion 37, the inner resin layer 19 inside is exposed.

  Next, as shown in FIG. 5C, the waveguide 9 is joined to the external resin layer removing portion 37 (arrow D in the figure). The waveguide 9 has a core portion 25 inside and a cladding portion 27 outside, and is separately molded by a molten resin drawing process or extrusion molding or injection molding in the same manner as a plastic fiber. The waveguide 9 and the well main body 35 are joined by fusion, for example. The waveguide 9 can be a multi-core plastic optical fiber. Here, as a multi-core type plastic optical fiber, for example, a double clad type multi-structure having a structure in which a first clad is thinly wrapped around a core in a ring shape, and a second clad is surrounded by the sea around the outer periphery. When a clad fiber is used, the loss is greatly reduced even when the fiber is sharply bent. At this time, when a single core type plastic fiber is used, the clad portion 27 may be slightly tapered in advance so that the core portion 25 protrudes from the tip of the waveguide 9. By doing in this way, the connection part of the well main body 35 and the waveguide 9 can be made smooth.

  As shown in FIG. 5D, the inner resin layer 19 and the core portion 25 are fused, and at the same time, the outer resin layer 23 and the cladding portion 27 are fused, whereby the well 3 is manufactured. In addition, if the core part 25 of the waveguide 9 and the internal resin layer 19 are optically joined, the joining method of the waveguide 9 and the well body is not limited to this. Further, the shape of the end of the waveguide 9 is not necessarily limited to this, and may be other shapes, or may be fused as it is without performing shape processing. Further, a protective covering material can be provided outside the cladding of the waveguide. In this case, the protective coating material is appropriately selected from polyethylene, flame retardant polyvinyl chloride, chlorinated polyethylene, PVC, nylon, fluorinated ethylene, polypropylene, and the like according to use conditions.

  Next, the temperature adjustment cycle of the reaction solution by the heater 7 will be described. FIG. 6 is a conceptual diagram showing a temperature cycle of PCR measurement. As described above, the amplification cycle 39 includes a denaturation step 41, an annealing step 43, and a polymerase extension step 45.

  The denaturation step 41 is a step of separating the template DNA into single strands at a temperature of about 95 ° C. The annealing step 43 is a step of lowering the temperature to about 50 ° C. and hybridizing the primer to DNA. The polymerase extension step 45 is a step of amplifying a DNA region sandwiched between primers by raising the temperature to about 70 ° C. and performing an enzymatic reaction of the DNA polymerase. The progress of the polymerase extension step can be grasped by real-time fluorescence measurement using the well 3 or the like described above. If it is determined by fluorescence measurement that the DNA region sandwiched between the primers has been amplified twice, the amplification cycle 39 can be further repeated to amplify the DNA.

  As described above, according to the present embodiment, since the heater 7 is disposed around the well 3, the temperature of the reaction solution 15 can be adjusted to a temperature suitable for each step. For this reason, the amplification cycle can be repeated only inside the well 3.

  Further, since the well 3 is formed in a two-layer structure having different refractive indexes, and the waveguide 9 is joined to the lower portion of the well 3, the light that has entered from the inside of the well 3 is confined in the well 3 and led out to the waveguide 9. can do. For this reason, even a very small amount of fluorescence can be detected with extremely high sensitivity as compared with the prior art.

  Further, since the reflection film 17 is provided on the upper end surface of the well, it is possible to prevent light from leaking out of the well. Depending on the shape of the well, the reflective film 17 forms a reflective film on the outer peripheral surface of the outer resin layer even when fluorescence leaks from the outer peripheral surface of the outer resin layer in the outer peripheral portion of the well at the portion with a large curvature at the well bottom. Thus, light can be prevented from leaking out of the well, and the degree of freedom in designing the curvature of the well bottom can be expanded.

  Next, another embodiment will be described. FIG. 7 is a schematic diagram showing a fluorescence measuring device 1a according to the second embodiment. In the following description, components having the same functions as those of the fluorescence measuring apparatus 1 are denoted by the same reference numerals as those in FIGS.

  The fluorescence measurement device 1a has substantially the same configuration as the fluorescence measurement device 1, but differs in that an optical switch 47 is used. A waveguide 9 from a plurality of wells is connected to one side of the optical switch 47. The optical filter 11 is connected to the other side of the optical switch 47, and the fluorescence measuring instrument 13 is disposed behind the optical filter 11.

  The optical switch 47 can switch light on the incident side. That is, any one of the waveguides 9a, 9b,..., 9n is optically connected to the optical filter 11 (fluorescence measuring device 13). Therefore, by switching the optical switch 47, the fluorescence in a plurality of wells can be measured with one optical filter 11 and the fluorescence measuring device 13.

  8 is a schematic diagram showing the configuration of the optical switch 47. FIG. 9A is a cross-sectional view taken along line EE in FIG. 8, and FIG. 9B is a cross-sectional view taken along line FF in FIG. The optical switch 47 mainly includes a motor 59, a pulley 61, a belt 63, a rotating body 49, a bearing 55, a fixed portion 51, and the like. The motor 59 is a drive unit that drives the rotating body via the pulley 61 and the belt 63. As the motor 59, a stepping motor can be used, but it is desirable to use a servo motor having a position feedback function.

  The pulley 61 is a part that transmits the power of the motor 59 to the belt 63. The belt 63 is preferably a toothed belt. In this case, a tooth shape corresponding to the toothed belt may be formed on the outer periphery of the pulley 61 and the rotating body 49 described later.

  The rotating body 49 is attached to a rotating shaft 53 provided at the center of the fixed portion 51 via a bearing 55. The fixing portion 51 is a portion that is not operated by the motor 59 and is fixed, and is arranged separately from the motor 59. This is because vibration from the motor is transmitted to the fixed portion when the fixed portion and the motor are brought into direct contact. The fixed portion 51 and the rotating body 49 are provided so as to face each other, and the rotating body 49 can rotate in the forward and reverse directions around an axis formed perpendicular to the surface of the fixed portion 51 facing the rotating body 49.

  In order to prevent the distance between the rotating body 49 and the fixed portion 51 from fluctuating, it is desired that the bearing 55 has a small displacement in the direction of the rotation axis. For example, an angular ball bearing can be used.

  Note that the drive of the rotating body 49 may not be a motor, and various devices such as an actuator can be applied. Further, for transmission of power between the drive unit and the rotating body, a gear or the like may be used instead of the belt 63. In any case, it is sufficient that the rotating body 49 can be rotated in the forward and reverse directions with respect to the fixed portion 51.

  As shown in FIG. 9A, a plurality of holes are formed on the fixed portion 51 side on a concentric circle having the same diameter around the rotation shaft 53 of the rotating body 49 with a predetermined interval. Further, the waveguide 9 is provided so as to penetrate the fixing portion 51. In the figure, for simplicity, three examples of waveguides 9a, 9b, and 9c are shown, but the number of waveguides that can be switched by the optical switch is not limited to this. The waveguides 9a, 9b, and 9c are inserted from the outer surface side of the fixed portion 51, and are arranged so that the end surfaces come to the surface positions on the surface facing the rotating body.

  Further, as shown in FIG. 9B, a fiber 57 is provided on the surface of the rotating body 49 facing the fixed portion 51. The fiber 57 is disposed so that the end face comes to the surface position facing the fixed portion 51, and further, at a position corresponding to the radial position of the waveguides 9a, 9b, 9c on the surface facing the fixed portion 51. Provided. That is, by rotating the rotating body 49 to a predetermined position, the end portion of the fiber 57 can be disposed opposite to the end portion position of the waveguides 9a, 9b, 9c.

  FIG. 10 is an enlarged cross-sectional view of a portion G in FIG. The end face of the fiber 57 is provided in the rotator 49 and is flush with the end face on the fixed portion side. Similarly, the end face of the waveguide 9a (9b, 9c) is disposed on the same plane as the end face on the rotating body side through the fixing portion 51. When the rotator 49 is rotated to a predetermined position, the waveguide 9a and the fiber 57 are positioned so as to face each other as shown in FIG. Further, since the gap 65 is formed between the rotating body 49 and the fixed portion 51, the rotating body 49 and the fixed portion 51 do not come into contact with each other when the rotating body 49 rotates. The same configuration may be adopted between the rotator 49 and the optical filter 11.

  When the operation of the rotator 49 is stopped at the position where the waveguide 9a and the fiber 57 face each other and fluorescence is sent from the waveguide 9a side (in the direction of arrow H in the figure), light enters from the end face of the fiber 57 (see FIG. Middle arrow I direction). That is, the waveguide 9a and the fiber 57 are optically connected.

  Note that the gap 65 is several tens of μm to several hundreds of μm, preferably about 10 to 100 μm, in view of the accuracy of members and operations.

  In order to prevent light leaking in the gap 65 from entering the other adjacent waveguides 9b and 9c without entering the fiber 57 at the position facing the waveguide 9a in the gap 65, the rotation is performed. It is desirable to apply a coating of an antireflection film that suppresses reflection of light, a pear treatment, or the like to the opposing surface of the body 49 and the fixing portion 51.

  As shown in FIG. 8, the fiber 57 is rotated from the position facing the waveguides 9 a, 9 b, 9 c on the surface facing the fixing portion 51, on the surface opposite to the surface facing the fixing portion 51. Up to the position, it is curved and built in a substantially S shape inside the rotating body 49. At each end of the fiber 57, the fiber 57 is disposed parallel to the rotational axis direction of the rotating body 49. Further, the radius of curvature of the fiber 57 inside the rotating body 49 is determined in consideration of the light transmission loss of the fiber 57. The radius of curvature of the fiber 57 is preferably a constant and smooth curve except for the straight portion.

  The optical filter 11 is provided at the approximate center of the surface of the rotating body 49 opposite to the surface facing the fixed portion 51. A fluorescence measuring device 13 is provided behind the optical filter 11. The end of the fiber 57 is optically connected to the optical filter 11. Therefore, the light introduced into the fiber 57 is measured by the fluorescence measuring instrument 13 through the optical filter 11.

  From this state, the rotating body 49 is rotated by a predetermined angle (arrangement angle of the waveguides 9a, 9b, 9c), and the corresponding waveguides 9a, 9b, 9c are sequentially stopped at the surface facing the fiber 57, By introducing light from each waveguide into the fiber 57, all the waveguides 9a, 9b, 9c can be optically connected to the fiber 57.

  As described above, according to the second embodiment, the same effect as that of the fluorescence measuring apparatus 1 can be obtained. Further, by using the optical switch 47, an expensive optical filter and a fluorescence measuring instrument are not required for each well, so that a compact and low-cost fluorescence measuring apparatus can be obtained.

  As mentioned above, although embodiment of this invention was described referring an accompanying drawing, the technical scope of this invention is not influenced by embodiment mentioned above. It is obvious for those skilled in the art that various modifications or modifications can be conceived within the scope of the technical idea described in the claims. It is understood that it belongs.

  For example, in the above-described embodiment, the method of separating, extracting and amplifying DNA in the reaction solution by the thermal cycle and measuring the fluorescence generated in the well using the heater 7 has been described. Not limited to. For example, without using a heater, it can also be used for fluorescence measurement of cells and the like and screening of cells using the same. Furthermore, it can be used for measurement of fluorescently labeled cells or chemically-stained cells, such as a scribing, even with an apparatus having no other heater according to the present invention.

DESCRIPTION OF SYMBOLS 1, 1a ......... Fluorescence measuring apparatus 3a, 3b, 3n ......... Well 5a, 5b, 5n ......... Light source 7 ......... Heaters 9a, 9b, 9n ......... Waveguides 11a, 11b, 11n ......... Optical filters 13a, 13b, 13n .... Fluorescence measuring instrument 15 .... Reaction solution 17 .... Reflection film 19 .... Internal resin layer 23 ...... Outer resin layer 25 ...... Core part 27 ....... Clad Section 29... Fluorescence generation center 31... Virtual light receiving surface 33... Receivable area 35... Well body 37 ... External resin layer removal section 39 Amplification cycle 41. ......... Annealing step 45 ......... Polymerase extension step 47 ......... Optical switch 49 ......... Rotating body 51 ......... Fixed part 53 ......... Rotating shaft 55 ......... Bearing 57 ......... Fiber 59 ......... Motor 61 ......... Pulley 63 ......... Belt 5 ......... gap 100 ......... template DNA
101a, 101b ......... Primer 103 ......... Hairpin primer 105 ......... Fluorescent dye 107 ......... Polymer 109 ......... Well 111 ......... Irradiation / light receiving portion 113 ......... Reaction solution 115 ......... Fluorescence generation center 117... Virtual light receiving surface 119...

Claims (5)

A fluorescence measurement PCR device comprising:
A well for holding the reaction solution;
A heater arranged on the outer periphery of the well and capable of adjusting the temperature of the reaction solution;
A light source for irradiating the reaction solution in the well with light;
A waveguide connected below the well;
A fluorescence measuring instrument for measuring fluorescence generated in the well,
The well has a two-layer structure of at least an outer resin layer and an inner resin layer,
The refractive index of the inner resin layer is larger than the refractive index of the outer resin layer,
The waveguide has a core portion made of the same material as the inner resin layer inside and a clad portion made of the same material as the outer resin layer inside, and the outer resin layer is removed at the lower portion of the well The core part is bonded to the exposed internal resin layer,
By adjusting the temperature by the heater, DNA can be separated, extracted and amplified in the reaction solution, and the fluorescence generated in the well is enclosed in the internal resin layer and led to the core part, Further, the fluorescence measuring apparatus can be measured by the fluorescence measuring instrument through an optical filter. A plurality of the wells are arranged,
The waveguide is connected to an optical switch, and the fluorescence from each of the wells can be measured by the fluorescence measuring device by switching fluorescence from each well by the optical switch. The fluorescence measuring apparatus as described.   The fluorescence measuring apparatus according to claim 1, wherein a reflective film is formed on at least one of the upper end surface of the well and the outer peripheral surface of the external resin layer.   The resin constituting the core of the well is any one of polycarbonate, modified polycarbonate, an acrylic polymer with a high Tg component monomer, a norbornene resin, and a silicone resin. The fluorescence measuring device according to any one of the above. A method for producing a well used in a fluorescence measurement PCR device,
The shape of the well body is molded so as to have a two-layer structure of an outer resin layer and an inner resin layer having a larger refractive index than the outer resin layer,
Removing the outer resin layer at the bottom of the well body to expose the inner resin layer;
Using a waveguide having a core portion made of the same material as the inner resin layer inside and a clad portion made of the same material as the outer resin layer outside,
A method for producing a well, comprising: bonding the core portion to the exposed inner resin layer; and bonding the clad portion to the outer resin layer.

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