JP2010276489A - Measuring device and measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve operability in total reflection illumination measurement by suppressing movement of an illumination domain generated, when replacing a substrate within a measuring field at the maximum, without having to perform optical adjustment. <P>SOLUTION: In a device, movement of an illumination domain in a measuring field caused by the thickness change of a substrate is removed by pressing the substrate onto a horizontal reference plane. Accordingly, the movement of the illumination domain in the measuring field caused by the thickness change of the substrate can be removed, and complex optical adjustment is not required; and a driving part, such as, an expensive XYZ-stage can be reduced. Consequently, operability of a total reflection illumination device is improved and thereby the cost of the device is reduced. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a total reflection illumination technique, for example, a technique for decoding a base sequence of a nucleic acid such as DNA or RNA.

The Human Genome Project, which invested 3 billion dollars between 1990 and 2005, could read the easiest part (93% of the total) to be read a few years earlier than planned, “Nature Reviews, Vol5, pp335, 2004 (Non-Patent Document 1) ”, the technology and methods necessary for decoding were left as a legacy. Such technology has been further improved since then, and today it is possible to perform genome decoding at an accuracy of about 20 million dollars ($ 2 × 10 ⁷ ). Nonetheless, at this amount, large-scale sequencing can only be done by specialized decoding centers or large research projects with large budgets. However, if the sequencing cost is reduced, a larger number of larger genomes can be handled. For example, it becomes possible to compare the genome of a patient and a healthy person, and as a result, improvement of the value of genome information can be expected. As seen in "From complete human genome decoding to" human "understanding, pp253, Shohei Hattori, Toyo Shoten, 2005" (Non-patent Document 2), acquisition of such basic data will develop into future tailor-made medicine. It is expected to contribute greatly to

Under the circumstances described above, two programs for “Innovative Genome Sequencing Technology” funded by the National Institutes of Health (NIH) will cost $ 100,000 for one human genome by 2009 ( $ 1 × 10 ⁵ ), and the goal is to make it $ 1000 ($ 1 × 10 ³ ) by 2014. This is the development of the so-called “$ 1000 genome” decoding technology.

Some next-generation sequencers have already been commercialized as seen in “Nature, vol.449, pp627, 2007 (Non-patent Document 3)”. These technologies have already achieved the cost of 1/10 to 1/100 of the prior art. In addition, the number of bases that can be measured by one analysis has reached 10 ⁹ orders. However, the improvement in cost of these sequencers has almost reached its limit, and it is expected that it will be difficult to achieve $ 1000 genome by using commercially available devices and systems.

Further, it takes the current although the next generation sequencers commercially available can identify the nucleotide sequence of up to about 10 ^9, only 2, 3 days or more time runtime sequencing of a large amount of data of about 10 ^9. In order to make genome sequence decoding at an individual level routine in medical practice, not only cost but also high-speed sequence decoding is required.

  One of the promising sequencing techniques expected in such a situation is the single molecule real-time sequencing method. This is a method that directly measures the actual site of enzyme reaction at the single molecule level. In the single molecule real-time sequencing method, fluorescence detection is performed in real time when the base is extended. Therefore, the speed of base sequence decoding is dramatically improved. In addition, single-molecule real-time measurement eliminates the need for amplification of a sample by PCR, and can greatly reduce the cost. Thus, single molecule real-time sequencing is one of the most promising technologies for achieving the $ 1000 genome.

  In “Next-Generateion Sequencing: Scientific and Commercial Implications of the $ 1,000 Genome, Kevin Davies, pp41, Insight Pharma Reports (Non-patent Document 4)”, a polymerase modified with a donor fluorophore is immobilized on a substrate as a single-molecule real-time measurement. The energy fluorescence transfer to the acceptor nucleotide accompanying the nucleic acid extension reaction is measured.

  Moreover, caged compounds can be mentioned as techniques for controlling various biological processes that proceed in cells and analyzing them in real time. A caged compound is a general term for compounds in which a bioactive molecule is modified with a photodegradable protecting group to temporarily lose its activity. It is named caged compounds in the sense that it is a molecule whose physiological activity is put in a cage and allowed to sleep. When a caged compound is irradiated with light having an optimum wavelength for the compound, the protecting group is removed only at the spot where the caged compound is irradiated, and the original physiological activity is expressed. In other words, it becomes possible to control chemical reactions by temporally and spatially dynamic light irradiation. In Japanese translations of PCT publication No. 2006-503586 (Patent Document 1) and US Patent Application Publication No. 2007/0059692 (Patent Document 2), caged compounds are used to create a pool of oligonucleotides. However, this method uses a sequential reaction, and is a method of sequentially decomposing a caged compound bonded to the terminal with the reaction. Therefore, this method is not used for real-time analysis. The target of this method is multi-molecule, not single molecule.

  Japanese Unexamined Patent Application Publication No. 2009-045057 (Patent Document 3) discloses a unimolecular sequence method using total reflection illumination measurement. Here, a laser is irradiated onto the substrate using a condensing lens and a prism, the laser is totally reflected on the substrate, and single molecules on the substrate are measured.

JP-T-2006-503586 US Patent Application Publication No. 2007/0059692 JP 2009-045057 A

Nature Reviews, vol5, pp335, 2004 From complete human genome decoding to understanding "human", pp253, Shohei Hattori, Toyo Shoten, 2005 Nature, vol.449, pp627, 2007 Next-Generateion Sequencing: Scientific and Commercial Implications of the $ 1,000 Genome, Kevin Davies, pp41, Insight Pharma Reports P.N.A.S. 2006, vol. 103, pp 19635-19640 P.N.A.S. 2008, vol. 105, pp 1176-1181

  As a result of intensive studies on the single molecule sequencing technique using total reflection illumination measurement, the present inventors have obtained the following knowledge.

  The problem of total reflection illumination measurement is poor operability. In the conventional total reflection illumination measurement, when the substrate to be totally reflected is replaced, there is a possibility that the illumination area moves due to a change in the thickness of the substrate. In this case, in order to perform normal measurement, it is necessary to adjust the prism and the condenser lens for total reflection illumination and move the moved illumination area again into the measurement visual field. If this adjustment is performed manually, the burden on the measurer is large. In particular, measurement is difficult for a measurer who is not skilled in the operation method of the apparatus. In order to overcome this problem, a method of adjusting the illumination area by using an XYZ stage can be considered. However, this method greatly increases the overall cost of the device. In addition, since the XYZ stage needs to periodically apply grease to the ball screw, the burden of maintenance also increases. In addition, the introduction of a drive part such as an XYZ stage has a risk of causing troubles such as failure. Therefore, there is a need for a total reflection illumination device that does not move the illumination area in the measurement field of view when the substrate is replaced without using a drive portion such as an XYZ stage.

  An object of the present invention relates to improving the operability in total reflection illumination measurement by suppressing the movement of the illumination area that occurs at the time of substrate replacement within the measurement field of view without optical adjustment.

  The present invention relates to removing a movement of an illumination region in a measurement visual field due to a change in thickness of the substrate by pressing the substrate against a horizontal reference plane.

  For example, a prism unit that integrally holds the prism and the substrate maintains a constant distance between the lower surface of the substrate and the upper surface of the prism, and holds the substrate and the prism in parallel. When measuring substrates with different thicknesses, the prism unit moves in the vertical direction by the thickness change of the substrate by pressing the prism unit holding the substrate against the reference surface. Since the laser beam is incident on the prism at 90 °, the laser beam does not depend on the vertical movement direction of the prism and travels straight without being refracted at the air-prism interface. As a result, even if the thickness of the substrate changes, the optical path of the laser becomes the same, and the movement of the illumination field within the measurement field can be eliminated. Preferably, the refractive indexes of the prism, the substrate, and the coupling agent are the same or substantially the same.

  According to the present invention, the movement of the illumination area in the measurement visual field due to the change in the thickness of the substrate can be eliminated, and complicated optical adjustment is not required, and the driving unit such as an expensive XYZ stage can be reduced. Therefore, the operability of the total reflection illumination device is improved, and the cost of the device can be reduced.

FIG. 3 is an explanatory diagram of a prism unit that integrally holds a prism and a substrate in Embodiment 1. FIG. 6 is an explanatory diagram of a method for mounting a substrate on a prism unit in Embodiment 2. FIG. 6 is an explanatory diagram of a prism unit that integrally holds a prism and a substrate in Embodiment 3. Explanatory drawing of the prism unit which integrally holds the prism and board | substrate in Example 4. FIG. FIG. 10 is an explanatory diagram of a prism unit that integrally holds a prism and a substrate in Embodiment 5. Explanatory drawing of 90 degrees incidence of the laser beam with respect to the prism in Example 6. FIG. FIG. 10 is an explanatory diagram of a nucleic acid analysis device in Example 7. FIG. 10 is an explanatory diagram of a nucleic acid analyzer in Example 7.

  In the embodiment, a substrate that can hold a sample, a prism, a light source, and a detector are provided, the substrate surface is pressed against a reference surface provided in the measurement device, the substrate is fixed, and the light from the light source via the prism is fixed. Disclosed is a measuring device that irradiates a substrate with light, causes total reflection on the surface of the substrate on which the sample is held, and detects light generated by the total reflection illumination with a detector.

  Further, in the embodiment, a substrate that can hold a sample, a prism, a light source, a detector, a reference surface that maintains a predetermined positional relationship with respect to the detector, and a substrate fixing device that presses the substrate surface against the reference surface A measuring device that irradiates light from the light source via a prism onto a substrate, causes total reflection on the surface of the substrate on which the sample is held, and detects light generated by the total reflection illumination by a detector. .

  In the embodiment, the substrate surface is pressed against a reference surface provided in the measurement device, the substrate capable of holding the sample is fixed to the measurement device, the light from the light source is irradiated to the substrate through the prism, and the sample is Disclosed is a measurement method in which light is generated by a total reflection on a held substrate surface and light generated by the total reflection illumination is detected by a detector.

  In the embodiment, a measuring device and a measuring method for pressing the prism against the substrate are disclosed. Further, it is disclosed that the substrate fixing device presses the prism against the substrate.

  In the embodiment, a measuring device and a measuring method for pressing a prism against a substrate through a liquid coupling agent are disclosed. Also disclosed is the presence of a liquid coupling agent between the prism and the substrate.

  In the embodiment, a measuring device and a measuring method for pressing the prism against the substrate using the restoring force of the elastic body are disclosed. Further, it is disclosed that the substrate fixing device includes an elastic body that presses the prism against the substrate.

  In the embodiment, a measuring device and a measuring method for pressing a prism against a substrate via a spacer are disclosed. It is also disclosed that a spacer exists between the prism and the substrate.

  In the embodiment, a measuring device and a measuring method for pressing a prism against a substrate via a solid coupling agent are disclosed. It is disclosed that there is a solid coupling agent between the prism and the substrate.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings. It should be noted that the embodiments can be combined as appropriate, and the combined form is also disclosed in this specification.

  A fluorescence measurement method using total reflection illumination using a reference surface will be described below with reference to FIG.

  In the total reflection illumination device of this embodiment, the phosphor 106 is fixed to a specific region on the upper surface of the substrate 105 having light transmittance. In order to measure the fluorescence emitted from the phosphor 106 by the total reflection illumination of the laser beam 104, the laser beam 104 is vertically incident on the prism 103, passes through the coupling agent 111 and the lower surface of the substrate 105, and is totally reflected on the upper surface of the substrate 105. Illuminate. The incident angle of the laser beam 104 on the upper surface of the substrate 105 is not less than the critical angle. The laser beam 104 excites the phosphor 106 on the upper surface of the substrate 105, and the generated fluorescence is condensed through the objective lens 101. In order to observe the target phosphor 106 in the measurement field, the phosphor 106 needs to be positioned at the intersection of the optical axis 112 of the objective lens 101 and the laser beam 104.

  However, the thickness of the different substrates 108 is not constant and varies. Therefore, when the phosphor 106 is observed with the substrate 105 replaced, the following problem occurs. For example, as shown in (1), when the substrate 108 after replacement becomes thicker than the substrate 105 before replacement, the illumination area where the total reflection illumination is generated is shifted to the left with respect to the optical axis 131. Similarly, when the thickness of the substrate 108 is thinner than the substrate 105, the illumination area is shifted to the right. When the thickness change of the substrate 105 is large, that is, when the movement of the illumination area is large, the illumination area moves out of the measurement visual field, and measurement becomes impossible. More specifically, if the change in the thickness of the substrate 105 is 50 μm and the incident angle of the laser beam 121 on the upper surface of the substrate 105 is 69 °, the illumination region moves by 50 μm × tan 69 ° = 135 μm. If the size of the measurement visual field is 100 μm square, the illumination area moves out of the measurement visual field, making measurement impossible. Although it is conceivable to suppress the change in thickness of the substrate as a method of preventing the movement of the illumination area, it is difficult to completely eliminate the thickness variation between different substrates at the μm order level, and it is a practical solution. is not.

  Further, as shown in (2), in the current total reflection illumination measurement, the prism 113 is washed for each measurement, and the prism is fixed to a predetermined prism holder. At that time, there is a possibility that the distance between the upper surface of the prism 113 and the lower surface of the substrate 114 is not constant. As a result, the thickness of the coupling agent 109 changes, and the optical path length through which the laser beam 116 passes changes. As a result, there arises a problem that the illumination area moves. In addition, as shown in (3), if the prism 110 is not held parallel to the substrate 117, the laser light is greatly refracted at the air-prism interface, and the illumination area moves. appear. The problems (2) and (3) are problems that occur because the prisms 109 and 110 and the substrates 114 and 117 are held independently.

  In the present embodiment, the problems occurring in the above (1), (2), and (3) are solved as follows. By pressing the substrate 105 against the horizontal reference plane 102, the movement of the illumination region due to the thickness change of the substrate 105 can be removed. More specifically, the distance between the lower surface of the substrate 105 and the upper surface of the prism 103 is kept constant by the prism unit 107 that integrally holds the prism 103 and the substrate 105, and the substrate 105 and the prism 103 are held in parallel. When measuring the substrates 105 having different thicknesses, the prism unit 107 holding the substrate 105 is pressed against the reference surface 102, whereby the prism position is moved in the vertical direction by the thickness change of the substrate. Since the laser beam 104 is incident on the prism 103 at 90 °, it travels straight without being refracted at the air-prism interface without depending on the vertical movement direction of the prism 103. As a result, even if the thickness of the substrate 105 changes, the optical path of the laser becomes the same, and the movement of the illumination field can be eliminated.

  According to the above-described procedure, for the thickness variation of the substrate 105 in (1), the upper surface of the substrate 105 is always positioned at the intersection of the optical axis 112 and the laser beam 104 by pressing the upper surface of the substrate 105 against the reference surface 102. Will come to be. Thereby, in the ideal state, the positional deviation of the illumination visual field does not occur. Further, even when a positional deviation occurs, it is possible to significantly reduce the positional deviation as compared with the case where there is no reference plane 102. Thereby, even when the substrate 105 is replaced, the illumination area remains at the same position in the measurement visual field, or the movement amount of the illumination area is reduced. Regarding (2), by using the prism unit 107 that holds the substrate 105 and the prism 103 together, the distance between the two can be kept constant. With regard to (3), it is possible to suppress the movement of the illumination area by using the prism unit 107 that holds the prism 110 and the substrate 117 with high parallelism.

  The refractive indexes of the substrate 105, the coupling agent 111, and the prism 103 are preferably the same or close to each other. As a specific example, the material of the substrate 105 and the prism 103 is synthetic quartz (refractive index 1.46), and the material of the coupling agent 111 is glycerol (refractive index 1.47). it can. If the refractive indexes are completely the same, no refraction occurs when the laser light 104 passes through the prism 103, the coupling agent 111, and the substrate 105, and the laser light 104 travels straight. Therefore, when the thickness of the substrate 105 increases, the thickness of the coupling agent 111 decreases, but the illumination area remains at the same position without being affected by these thickness changes.

  The incident angle of the laser beam 104 with respect to the prism 103 is 90 °. The reason is that, as will be described later in detail, it is possible to eliminate the influence of the horizontal displacement of the prism and the vertical displacement of the prism 103 by 90 ° incidence.

  In this embodiment, a substrate mounting method in a total reflection illumination device using a reference surface will be described with reference to FIG. Hereinafter, the difference from the first embodiment will be mainly described.

  In order to remove or suppress the movement of the illumination area when the substrate is replaced, it is necessary to match the positions of the intersection 205 where the optical axis 203 of the objective lens 201 and the laser beam 204 intersect with the phosphor 209 on the substrate 208. is there.

  In order to achieve this, a coupling agent 207 is dropped on the prism 206 and the substrate 208 is placed on the prism unit 210. Thereby, the space between the substrate 208 and the prism 206 is filled with the coupling agent 207. The substrate 208 and the prism 206 are held in an integrated state by the prism unit 210. The prism unit 210 is moved upward and pressed against the reference surface 202. The reference surface 202 is in a horizontal state with high accuracy. Thereby, the position of the phosphor 209 on the substrate 208 can be matched with the intersection 205. Accordingly, in the ideal state, the illumination area does not move within the measurement visual field. In actual measurement, it is possible to suppress the movement of the illumination area.

  In this embodiment, a prism unit having a mechanism for pressing a substrate will be described with reference to FIG. Hereinafter, the difference from the first and second embodiments will be mainly described.

  In the present embodiment, unlike the second embodiment, a mechanism 301 for pressing the prism 302 against the substrate 305 exists under the prism 302. The mechanism 301 presses the substrate 305 using a spring or a sponge. The substrate 301 is pressed against the reference surface 304 via the prism 302 by the mechanism 301. Since the reference surface 304 has a high degree of parallelism, the parallelism tolerance of the prism 302 can be removed by pressing the mechanism 301. As a result, the substrate 305 and the prism 302 also achieve high parallelism. Note that the coupling agent 303 between the substrate 305 and the prism 302 spreads at the boundary between the prism 302 and the substrate 305 due to the pressing of the prism 302. Therefore, it is desirable that the amount of the coupling agent 303 is very small. A major difference from the second embodiment is that the distance between the prism 302 and the substrate 305 is almost zero by the pressing of the mechanism 301.

  In this embodiment, a prism unit having a mechanism for pressing the substrate and a spacer for maintaining a distance between the substrate and the prism will be described with reference to FIG. Hereinafter, it demonstrates centering on difference with Examples 1-3.

  In the present embodiment, as in the third embodiment, a mechanism 403 for pressing the prism 402 against the substrate 405 exists below the prism 402. The substrate 405 is pressed against the reference surface 404 by the mechanism 403 via the prism 402 and the spacer 401. Since the reference surface 404 has a high degree of parallelism, the substrate 405 and the prism 402 also achieve a high degree of parallelism by pressing the mechanism 403. Note that the distance between the substrate 405 and the prism 402 is kept constant because the spacer 401 exists. The feature of this embodiment is that, while the distance between the substrate 405 and the prism 402 is kept constant by the spacer 401, the substrate 405 and the prism 402 are highly parallel to the reference surface 404 by pressing the mechanism 403.

  In this embodiment, a prism unit in which a coupling agent that is a normal solution is replaced with a solid silicon rubber will be described with reference to FIG. Hereinafter, it demonstrates centering around difference with Examples 1-4.

  As the coupling agent, a liquid such as glycerol is usually used, and these coupling agents are liquids. Therefore, the coupling agent may flow out to the incident surface of the prism 503 where the laser beam 504 is incident. In that case, it is necessary to remove the prism 503 from the prism unit 505, wash away the coupling agent with ultrapure water, dry the prism 503, and attach the prism 503 to the prism unit 505 again. This operation is complicated and also requires adjustment to bring the illumination area within the field of view. These operations significantly reduce operability. In the present embodiment, a solid silicon rubber 501 is used as a substitute for the liquid coupling agent. Similar to the above-described embodiment, by pressing the prism unit 505 upward against the substrate 502, measurement can be performed without moving the illumination area. The advantage of this embodiment is that the operability can be greatly improved by using the solid silicon rubber 501 as a substitute for the liquid coupling.

  In this embodiment, the vertical incidence of laser light on the prism will be described with reference to FIG. Hereinafter, it demonstrates centering around difference with Examples 1-5.

  As shown in (1), the laser beam 605 is perpendicularly incident on the incident surface of the prism 601 at an angle of 90 °. Even if the prism 601 is displaced in the horizontal direction from this state, since the refractive indexes of the prism 601, the coupling agent 602, and the substrate 603 are constant, the laser light 605 travels straight. Therefore, even when a positional shift occurs, the phosphor 604 stays at the same position in the measurement visual field, and the illumination visual field does not move. Similarly, in (2), the prism 611 is displaced in the vertical direction. Even in this case, since the laser beam 615 goes straight, the illumination area in the measurement visual field does not move. Therefore, stable measurement can be performed without being affected by the positional deviation of the prism in the horizontal and vertical directions.

  In this example, a nucleic acid analysis device and a nucleic acid analysis apparatus will be described. Hereinafter, it demonstrates centering around difference with Examples 1-6.

  FIG. 7 shows an outline of the nucleic acid analysis device. A plurality of regions 702 in which metal bodies are arranged in a lattice pattern are mounted on the support base 701. The metal body corresponds to a structure in which a probe is fixed between two adjacent metal bodies. The arrangement interval can be appropriately set according to the specifications of the nucleic acid sample to be analyzed and the fluorescence detection apparatus. For example, if a glass substrate having a size of 25 mm × 75 mm is used as a support base 501 and a region 702 in which metal structures are arranged in a grid at intervals of 1 micrometer is 5 mm × 8 mm, 40 million kinds of nucleic acid molecules can be analyzed per region. About eight regions can be mounted on the support base 701. Therefore, for example, when used for RNA expression analysis, since about 400,000 molecules of RNA are expressed per cell, RNA expression frequency analysis can be performed sufficiently accurately as in digital counting, About 8 analyzes can be performed on one substrate.

  In this embodiment, a plurality of reaction regions are provided on the support substrate 501 by pressing a light-transmitting support substrate 701 against a reaction chamber 703 provided with a channel 704 in advance. In the reaction chamber 703, a groove of the flow path 704 is dug in advance, and the reaction chamber 703 is used by being bonded to the device. More specifically, a temperature control unit 705 for storing / controlling the temperature of nucleic acid samples, reactive enzymes, buffers, nucleotide substrates, etc., a dispensing unit 706 for sending out the reaction liquid, a valve 707 for controlling the liquid flow, and a waste liquid tank 708 Composed. If necessary, install a temperature controller to control the temperature. By supplying a cleaning liquid through the channel 704 before starting the reaction, the support substrate can be cleaned. The cleaning liquid from which the support substrate has been removed is stored in a waste liquid tank 708.

  FIG. 8 shows an outline of a nucleic acid analyzer to which a nucleic acid analysis device is attached. In this apparatus, means for supplying one or more types of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthase, primers, and nucleic acid samples to the nucleic acid analysis device, and irradiating the nucleic acid analysis device with light And fluorescence detection means for measuring the fluorescence of the fluorescent dye incorporated into the nucleic acid chain by the nucleic acid extension reaction caused by the nucleotide, the nucleic acid synthesizing enzyme, and the nucleic acid sample coexisting on the nucleic acid analysis device; Is provided. More specifically, the nucleic acid analysis device 805 is installed in a reaction chamber composed of a cover plate 801, a detection window 802, an inlet 803 that is a solution exchange port, and an outlet 804. Note that the thickness of the detection window 802 is 0.17 mm. Laser light 808 and 809 oscillated from a YAG laser light source (wavelength 532 nm, output 20 mW) 806 and a YAG laser light source (wavelength 355 nm, output 20 mW) 807 are circularly polarized only by the λ / 4 plate 810, and the laser light 809 is circularly polarized. The two laser beams are adjusted to be coaxial by a mirror 811 (reflects 410 nm or less), then condensed by a lens 812, and then irradiated to a nucleic acid analysis device 805 through a prism 813 at a critical angle or more. The phosphor captured during the extension reaction is excited by the laser light, and part of the enhanced fluorescence is emitted through the detection window 802. Further, the fluorescence emitted from the detection window 802 is converted into a parallel light beam by the objective lens 814 (× 60, NA 1.35, working distance 0.15 mm), and background light and excitation light are blocked by the optical filter 815, thereby forming an image. The image is formed on the two-dimensional CCD camera 817 by the lens 816.

  The support substrate 701 is not particularly limited, and one or more materials selected from plastics, inorganic polymers, metals, natural polymers, and ceramics can be used. Examples of the plastic include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenol resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyvinyl fluoride, polyimide, and acrylic resin. Examples of the inorganic polymer include glass, quartz, carbon, silica gel, and graphite. Examples of the metal include room temperature solid metals such as gold, platinum, silver, copper, iron, aluminum, and magnets. Examples of the ceramic include sapphire, alumina, silica, silicon carbide, silicon nitride, and boron carbide. In particular, quartz, quartz, various optical glasses such as BK-7 and SF-2, sapphire, etc., which are excellent in light transmissivity, have a high flatness at the substrate interface and can easily maintain the flatness, are preferable.

  Further, an appropriate functional group may be introduced on the surface of the support substrate 701 so that a primer such as a primer or nucleic acid synthesis can be easily fixed. Examples of such functional groups include amino groups, thiol groups, carboxyl groups, hydroxyl groups, aldehyde groups, and ketone groups. Examples of a method for introducing these functional groups include a method in which an appropriate oxide is introduced onto the support substrate 701 and then reacted with a carboxylic acid, a phosphonic acid, a phosphate ester, and an organosilane compound. Moreover, after introducing a noble metal such as gold, silver, mercury, indium, palladium, ruthenium, or zinc on the surface of the support, an organic sulfur compound, an organic selenium compound, or an organic tellurium compound is reacted. . Furthermore, as a technique for improving the reaction efficiency for immobilizing the probe, a functional group such as NHS-ester group, imide ester group, sulfidyl group, epoxy group, hydrazide group, etc. may be introduced using a divalent compound. good. By supplying a probe modified with an amino group, a thiol group, a biotin group or the like to the functional group introduced on the outermost surface of the support, the probe can be effectively immobilized on the support substrate 701. it can. Alternatively, the probe may be immobilized via a compound such as avidin, dendron, or crown ether.

  The probe is not particularly limited as long as it can supplement the nucleic acid sample to be measured. Examples of probes that can directly capture a nucleic acid sample include nucleic acids such as DNA, RNA, and PNA, and proteins such as enzymes. Alternatively, a nucleic acid sample may be captured via a chromosome, nucleoid, cell membrane, cell wall, virus, antigen, antibody, lectin, hapten, receptor, peptide, sphingosaccharide, sphingolipid, and the like.

  In the case of the sequential reaction method, as a nucleotide with a fluorescent dye, there is a 3 at the 3′OH position of ribose as disclosed in “PNAS 2006, vol. 103, pp 19635-19640 (Non-patent Document 5)”. A '-O-allyl group can be used as a protecting group, and it can be linked to a fluorescent dye via an allyl group at the 5-position of pyrimidine or at the 7-position of purine. Since the allyl group is cleaved by light irradiation or contact with palladium, the quenching of the dye and the control of the extension reaction can be achieved simultaneously. Even in the sequential reaction, it is not necessary to remove unreacted nucleotides by washing.

  Further, in this example, since the washing step is not necessary as disclosed in “PNAS 2008, vol. 105, pp 1176-1181” (non-patent document 6), the extension reaction can be measured in real time. Is possible. As described above, by assembling a nucleic acid analyzer using the nucleic acid analysis device of this embodiment, the analysis time can be shortened and the device and the analyzer can be simplified without using a washing step. In addition to the sequential reaction method, it is also possible to measure the base extension reaction in real time, which can greatly improve the throughput compared to the conventional technique.

101, 201, 814 Objective lens 102, 202, 304, 404 Reference plane 103, 206, 302, 402, 601, 813 Prism 104, 116, 121, 204, 605, 808, 809 Laser light 105, 108, 114, 117 , 208, 305, 405, 502, 603 Substrate 106, 209, 604, 614 Phosphor 107, 210, 306, 406, 505 Prism unit 111, 207, 303, 407, 602, 612 Coupling agent 112, 131, 203 Optical axis 115 of objective lens 115 Distance between substrate and prism 205 Intersection point 702 of laser beam and optical axis of objective lens 703 Reaction chamber 704 Flow path 705 Temperature control unit 706 Dispensing unit 707 Valve 708 Waste liquid tank 801 Cover plate 802 Detection window 80 Nucleic inlet 804 outlet 805 the analysis device 806 YAG laser light source (wavelength 532 nm, output 20 mW)
807 YAG laser light source (wavelength 355 nm, output 20 mW)
810 λ / 4 plate 811 Dichroic mirror 812 Lens 815 Optical filter 816 Imaging lens 817 Two-dimensional CCD camera

Claims (18)

A substrate capable of holding a sample, a prism, a light source, and a detector, irradiating the substrate with light from the light source via the prism, totally reflecting on the substrate surface holding the sample, and In the measuring device for detecting the light generated by the total reflection illumination by the detector,
A measuring apparatus, wherein the substrate surface is fixed by pressing the substrate surface against a reference surface provided in the measuring apparatus. The measuring device according to claim 1,
A measuring apparatus, wherein the prism is pressed against the substrate. The measuring device according to claim 1,
A measuring apparatus, wherein the prism is pressed against the substrate through a liquid coupling agent. The measuring device according to claim 1,
A measuring apparatus, wherein the prism is pressed against the substrate by using a restoring force of an elastic body. The measuring device according to claim 1,
A measuring apparatus, wherein the prism is pressed against the substrate through a spacer. The measuring device according to claim 1,
A measuring apparatus, wherein the prism is pressed against the substrate through a solid coupling agent. A substrate capable of holding a sample, a prism, a light source, and a detector, irradiating the substrate with light from the light source via the prism, totally reflecting on the substrate surface holding the sample, and In the measuring device for detecting the light generated by the total reflection illumination by the detector,
A reference plane that maintains a predetermined positional relationship with the detector;
And a substrate fixing device that presses the substrate surface against the reference surface. The measuring device according to claim 7, wherein
The measurement apparatus, wherein the substrate fixing tool presses the prism against the substrate. The measuring device according to claim 7, wherein
A measuring apparatus, wherein a liquid coupling agent is present between the prism and the substrate. The measuring device according to claim 7, wherein
The measurement apparatus, wherein the substrate fixing device includes an elastic body that presses the prism against the substrate. The measuring device according to claim 7, wherein
A measuring apparatus comprising a spacer between the prism and the substrate. The measuring device according to claim 7, wherein
A measuring device, wherein a solid coupling agent is present between the prism and the substrate. Press the substrate surface against the reference surface provided in the measurement device, and fix the substrate that can hold the sample to the measurement device.
The substrate is irradiated with light from a light source through a prism, and is totally reflected on the substrate surface on which the sample is held,
A measurement method in which light generated by the total reflection illumination is detected by a detector. The measurement method according to claim 13,
A measuring method comprising pressing the prism against the substrate. The measurement method according to claim 13,
A measuring method, wherein the prism is pressed against the substrate through a liquid coupling agent. The measurement method according to claim 13,
A measuring method comprising pressing the prism against the substrate by using a restoring force of an elastic body. The measurement method according to claim 13,
A measuring method, wherein the prism is pressed against the substrate through a spacer. The measurement method according to claim 13,
A measuring method, wherein the prism is pressed against the substrate through a solid coupling agent.

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