Abstract:
Provided is a reaction device for nucleic acid analysis wherein microparticles, which carry a nucleic acid to be detected having been immobilized thereon, are aligned in a lattice form on a substrate according to the pixel size of a two-dimensional sensor. By this reaction device for nucleic acid analysis which is provided with a channel-forming reaction chamber on the substrate ( 101 ), the nucleic acid having been immobilized on the microparticles ( 103 ) on the substrate ( 101 ) is detected. The microparticles ( 103 ), which carry the nucleic acid to be detected having been immobilized thereon, are arranged by microstructures ( 102 ) aligned on the substrate ( 101 ).

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a nucleic acid analyzer, a reaction device for nucleic acid analysis, and a substrate of a reaction device for nucleic acid analysis. 
       BACKGROUND ART 
       [0002]    New technologies have been developed to determine base sequences of DNA&#39;s and RNA&#39;s. 
         [0003]    In a method utilizing electrophoresis, which is usually used at present, a cDNA fragment sample, which is synthesized in advance from a DNA fragment or an RNA sample for sequence determination by performing a reverse transcription reaction, is prepared, a dideoxy reaction is performed by the well-known Sanger method, and then electrophoresis is performed to measure and analyze a pattern of separated base ladders. 
         [0004]    On the other hand, in recent years, a method has been proposed for immobilizing many DNA fragments as samples on a substrate to determine sequence information of many fragments in parallel. 
         [0005]    In Non-Patent Literature 1, microparticles are used as carriers for supporting DNA fragments to perform PCR&#39;s on the microparticles. After that, the microparticles supporting PCR-amplified DNA fragments are put into a plate provided with many holes, a diameter of which is matched to a size of the microparticles, to read out by a pyrosequence method. 
         [0006]    Also, in Non-Patent Literature 2, using the microparticles as supports for supporting DNA fragments, PCR&#39;s are performed on the microparticles. After that, the microparticles are scattered and immobilized on a glass substrate, enzyme reactions (ligations) are performed on the glass substrate to let substrates with fluorescent dyes to be incorporated, and sequence information of each fragment is obtained by performing fluorescence detection. 
         [0007]    Further, in Non-Patent Literature 3, many DNA probes having the same sequence have been immobilized on a substrate. Also, after scission of a DNA sample, an adapter sequence of a strand complementary to the DNA probe sequence is added to the terminal of each DNA sample fragment. By subjecting these to hybridization on the substrate, the sample DNA fragments are immobilized one molecule by one molecule randomly on the substrate. In this case, after performing DNA elongation on the substrate to let substrates with fluorescent dyes to be incorporated, washing off of unreacted substrates and fluorescence detection are preformed so that sequence information of sample DNA&#39;s is acquired. 
         [0008]    As described above, a method for immobilizing many DNA fragment samples on a substrate and determining sequence information of many fragments in parallel has been developed and been put into practical use. 
       CITATION LIST 
     Non Patent Literature 
       [0009]    NON PATENT LITERATURE 1: Nature 2005, Vol. 437, pp. 376-380. 
         [0010]    NON PATENT LITERATURE 2: Science 2005, Vol. 309, pp. 1728-1732. 
         [0011]    NON PATENT LITERATURE 3: Science 2008, Vol. 320, pp. 106-109. 
         [0012]    NON PATENT LITERATURE 4: P.N.A.S. 2006, Vol. 103, pp. 19635-19640. 
         [0013]    NON PATENT LITERATURE 5: P.N.A.S. 2008, Vol. 105, pp. 1176-1181. 
         [0014]    NON PATENT LITERATURE 6: Anal. Biochem. 2003, Vol. 320, pp 55-65. 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0015]    However, even when parallel analysis methods as above are used, several days are required to analyze all human genes, and thus an analysis method having further higher throughput has been desired. A plate shown in Non-Patent Literature 1 has a diameter of a hole thereon of 44 μm, and a diameter of the microparticles is 22 μm. There is a problem that density of particles on the plate is low and, thus, the number of the DNA fragments which can be analyzed at once is small. The diameter of the microparticles of Non-Patent Literature 2 is as small as 1 μm and density of the microparticles on the substrate is high. However, because the microparticles are immobilized randomly on the substrate, it is necessary to use a two-dimensional sensor having many numbers of pixels in order to isolate and detect fluorescence from beads which are close together. It has problems of longer data transmission time and decreased throughput of analysis because of increase in the number of data per analysis. In addition, there is also a problem that a detection apparatus becomes expensive because a condensing lens of a large numerical aperture, NA, is necessary. A plurality of reaction solutions are sent to perform ligation reactions and it also causes a problem that the beads are peeled off from the substrate by resistance at the time of sending solutions. Also in Non-Patent Literature 3, arrangement of the DNA sample fragments of measurement objects is random. There is a problem of low throughput and an expensive detection apparatus, similar to in Non-Patent Literature 2. 
       Solution to Problem 
       [0016]    As result of intensive study the inventors of the present invention have completed development of a reaction device for nucleic acid analysis detectable with a two-dimensional sensor having a small number of pixels while yielding high throughput. 
         [0017]    This reaction device for nucleic acid analysis is provided with a substrate and a reaction chamber forming a flow channel on the substrate, and is a reaction device for nucleic acid analysis which detects nucleic acids immobilized on carriers on the substrate, wherein microstructures are arranged regularly on the substrate, and wherein each carrier is immobilized by the microstructures. 
         [0018]    By the carriers being immobilized by the microstructures arranged regularly, it becomes possible to arrange the immobilized carriers regularly. 
         [0019]    Therefore, even when a two-dimensional sensor having a small number of pixels is used, it becomes possible to isolate and detect fluorescence from beads which are close together. Accordingly, since the number of data per analysis decreases, the data transmission time becomes shorter and throughput of analysis is increased. Further, there is no need to use a condensing lens with a large numerical aperture, NA, and the detection device can be made inexpensive. 
         [0020]    The present invention is a nucleic acid analyzer comprising a reaction device for nucleic acid analysis with carriers on which nucleic acids to be detected are immobilized arranged regularly on the substrate, an irradiation light source, and a detection unit including a two-dimensional sensor, and it is characterized to include microstructures on the substrate. By giving certain regularity in arrangement or shapes of these microstructures, positions of the carriers can be controlled. 
         [0021]    In addition, when the carriers are the microparticles, it is possible to arrange them in high density because their particle diameters are small, and, together, the amount of nucleic acid immobilized on the microparticles can be increased because the shapes of the particles are large in the surface area. 
         [0022]    Also, by making the microstructures shorter in the length in a longer direction when viewed from above than the carriers, the microparticles can be arranged in high density. 
         [0023]    Further, each one of the above-described microparticles may be present as being surrounded by the microstructures. When a solution containing a reagent is sent onto a substrate on which the microparticles are immobilized, peeling of the carriers (for example, beads) during sending the solution can be prevented directly by contact with the microstructures or indirectly via control of water flow. 
         [0024]    Furthermore, by arranging the microstructures in a lattice form, the microparticles can be squarely arranged two-dimensionally in high density conforming with pixel sizes of a two-dimensional sensor. 
         [0025]    Besides, because the microparticles are made of a magnetic material, the microparticles can be immobilized on the substrate using magnetic force. 
       Advantageous Effects of Invention 
       [0026]    Using the microstructures arranged along with the pixels of the two-dimensional sensor, the microparticles on which DNA&#39;s to be detected are immobilized are immobilized on the substrate. Detection by a two-dimensional sensor having a small number of pixels is enabled. Nucleic acid sequence can be analyzed with high throughput by decreasing the number of data in detection. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0027]      FIG. 1  is a drawing for explaining one example of a configuration of a reaction device for nucleic acid analysis of the present invention. 
           [0028]      FIG. 2  is a drawing for explaining one example of a configuration of a reaction device for nucleic acid analysis of the present invention. 
           [0029]      FIG. 3  is a drawing for explaining one example of a manufacturing method of a reaction device for nucleic acid analysis of the present invention. 
           [0030]      FIG. 4  is a drawing for explaining another variation of microstructures in a device for nucleic acid analysis of the present invention. 
           [0031]      FIG. 5  is a drawing for explaining another variation of microstructures in a device for nucleic acid analysis of the present invention. 
           [0032]      FIG. 6  is a drawing for explaining another variation of microstructures in a device for nucleic acid analysis of the present invention. 
           [0033]      FIG. 7  is a drawing for explaining one example of a configuration of a reaction apparatus for nucleic acid analysis of the present invention. 
           [0034]      FIG. 8  is a drawing for explaining one example of a configuration of a reaction apparatus for nucleic acid analysis of the present invention. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0035]    Explanation is given below on an embodiment of the present invention with reference to drawings. 
         [0036]    (Regarding a Substrate and Microparticles Used for a Reaction Device for Nucleic Acid Analysis) 
         [0037]    Explanation is given on a substrate for a reaction device for nucleic acid analysis of the present invention with reference to  FIG. 1 . Microstructures  102  are arranged on a substrate  101  in a lattice form (the upper left drawing of  FIG. 1 ). Onto the substrate  101  a solution  104  (the upper right drawing of  FIG. 1 ) is sent, which contains the microparticles  103  (which may also be called carriers, or beads) on which nucleic acids to be detected are immobilized. By making the microstructures  102  and the microparticles  103  interact, the microparticles  103  are arranged on the substrate in a lattice form (the lower drawing of  FIG. 1 ). 
         [0038]    As the substrate  101 , a substrate made of an inorganic substance such as a glass substrate, a sapphire substrate, and a silicon substrate, a substrate made of a metal such as stainless steel, a substrate made of an organic substance such as a polymethylmethacrylate resin, a polycarbonate resin, and a cycloolefin resin, can be used. 
         [0039]    As shapes of the microstructures  102 , various shapes such as a cylinder, a cone, a triangle pole, a triangular pyramid, and a quadrangular pole can be used. Generally, variations in the shapes or the sizes of these microstructures  102  arise in production even in the same plane. It is preferable that the structures are provided with tapers so that it is possible to contact with the microparticles  103  at any of the positions even when the shapes or the sizes of a plurality of microstructures  102  in contact with one microparticle  103  vary. It should be noted that “tapers” here means such inclinations of the microstructures as becoming thinner from a closer side of the substrate to a further side. 
         [0040]    A material of the microstructures  102  is not especially limited, as long as it can be finely processed to equal to or smaller than the diameter of the microparticles  103 . As a combination of such a material and a processing method, a combined processing of photolithography and dry etching in silicon, nanoimprint lithography in a polymethylmethacrylate resin or a cycloolefin resin; or the like can be listed. 
         [0041]    The microstructures  102  and the microparticles  103  are immobilized via various bondings. As such bonding schemes, hydrophobic bonding, electrostatic interaction, covalent bonding, and the like can be used. If it is electrostatic interaction, by introducing amino groups on the surfaces of the microstructures, the microparticles  103  adsorbing DNA&#39;s efficiently can be immobilized. Also, if it is covalent bonding, by introducing in advance amino groups or sulfide groups at the terminals of immobilized DNA&#39;s of the microparticles  103 , for example, and making reaction with functional groups on the surfaces of the substrate  101  and the microstructures  102  via linker molecules, firm bonding can be attained. By using such chemical bonding, peeling or dropping of the microparticles  103  in sending a solution for washing or the like can be prevented. 
         [0042]    In introduction of functional groups onto the substrate surface, it is necessary to select a suitable method in consideration of a material thereof; for example, when the material is an organic resin, introduction of hydroxyl groups or carboxyl groups by oxidation treatment, graft polymerization of monomer molecules having desired functional groups, or the like can be listed. 
         [0043]    As a linker molecule, although it should be selected in consideration of combination of functional groups of the substrate  101  and the DNA terminals, for example, a molecule having a sulfohydryl group, an amino group, a carboxyl group, a phosphate group, an aldehyde group, or the like can be used. Also, functional groups on the surfaces of the microparticles in addition to functional groups at the DNA terminals can also be utilized. For example, in many cases the surfaces of the microparticles have carboxyl groups to enhance dispersibility, and these carboxyl groups and carboxyl groups generated on the substrate surfaces by oxidation treatment or graft polymerization of an organic substrate can be immobilized using molecules having a plurality of amino groups, represented by polyamine, and a reagent such as carbodiimide. In addition, a metal ion can be used as a linker. For example, a tetravalent Zr ion has been known to exhibit interactions with a carboxyl group and a single-stranded DNA; the tetravalent Zr ion is immobilized using a carboxyl group generated on the surface by, for example, oxidation treatment or graft polymerization of the surface of an organic substrate so that immobilization is possible using interaction of the Zr ion thereof and a single-stranded DNA. 
         [0044]    Incidentally, use of such a linker is not essential, and it is possible to bond the substrate  101  and the microparticles  103 , for example, via a carboxyl group generated by oxidation of an organic substrate, and an amino group introduced at the DNA terminal on the surfaces of the microparticles. As the microparticles  103 , those having a diameter of 5 nm to 100 nm are available on the market, and they can be utilized. A material composing the microparticles includes polystyrene, a magnetic material represented by iron oxide, semiconductor microparticles, or the like, and the magnetic material, where magnetic force can be utilized for alignment, is particularly preferable. 
         [0045]    As a method for forming the microstructures  102  on the substrate  101 , when a smooth material is an inorganic material, thin-film processing, which is already put into practice for a semiconductor, may be utilized. For example, it can be manufactured by vapor deposition or sputtering deposition through a mask, or dry or wet etching after formation of a thin film by vapor deposition or sputtering deposition. On the other hand, when a material of the substrate  101  is an organic material such as PMMA, a molding scheme like nanoimprint may also be used. 
         [0046]    (Regarding a Reaction Device for Nucleic Acid Analysis) 
         [0047]    Explanation is given on an example of a preferable configuration of a reaction device for nucleic acid analysis of the present invention with reference to  FIG. 2 . On a substrate  201 , a plurality of regions  202  are mounted, where the microparticles (not shown) are arranged in a lattice form. Spacings of the regions  202  can be set properly according to a nucleic acid sample to be analyzed and specifications of a fluorescence detection device. Installation of a plurality of reaction regions on the substrate  201  can be attained by covering a reaction chamber  204  provided with flow channels  203  in advance on the optically transparent substrate  201 . The reaction chamber  204  is made of a base substance of a resin such as PDMS (polydimethylsiloxane) on which grooves of the channels  203  are excavated in advance to form channels, and is used as being pasted together on the substrate  201 . The reaction device for nucleic acid analysis produced by pasting can be used together with a temperature control unit  205  for storing and managing temperature of a nucleic acid sample, a reaction enzyme, a buffer, a nucleotide matrix, or the like, a dispensing unit  206  for sending a reaction solution out, valves  207  for controlling liquid flows, and a waste liquid tank  208 . Temperature is controlled by arranging a temperature controller as needed. After completion of a reaction, a cleaning solution is supplied through the channels  203  and stored in a waste liquid tank  208 . 
         [0048]    (A Manufacturing Method of a Substrate for the Reaction Device for Nucleic Acid Analysis) 
         [0049]    Explanation is given on a manufacturing method of a substrate for the reaction device for nucleic acid analysis with reference to  FIG. 3 . On a substrate  501  made of a thermoplastic resin such as a cycloolefin resin (Zeonor 1060R manufactured by Zeon Corp.) microstructures  503  are produced using a stamper  502 . The substrate  501  having the microstructures  503  is obtained by a nanoimprint lithography method, in which the stamper  502  is pressed against the substrate  501  heated up to glass transition temperature or higher. In this case, the stamper  502  has a depression or a protrusion of a cross hair or a marker like a cross hair except for missing its center portion, and a marker  504  is also formed simultaneously on the substrate  501  having the microstructures  503 . By producing this marker  504 , arrangement positions and angles of the microstructures  503  can be estimated to estimate final positions of the microparticles, even from a low magnification image in which the microstructures  503  can not be directly observed. 
         [0050]    Incidentally, the explanation was given on an example where the microstructures are arranged in a lattice form in  FIG. 3 . An arrangement method is not limited to this, however, and it can be varied freely to match with arrangement of CCD elements. For example, when a rectangular pixels CCD is used, in which arrangement intervals of light receiving elements composing the CCD are different in a longitudinal direction and a lateral direction, the microstructures may be arranged with different intervals in a longitudinal direction and a lateral direction in agreement with arrangement intervals of the light receiving elements. Also, when it is a CCD with its light receiving elements being in a honeycomb structure, for example, it may be arranged in a square lattice form inclined by 45 degrees from the arrangement angle of the CCD. 
         [0051]    Further, not being limited to line up the microstructures in a constant interval, such an arrangement may also be adopted that, for example, an interval between the first line and the second line is short, an interval to the next third line is long, and an interval to the fourth line is short again. 
         [0052]    (Regarding Other Variations of the Substrate for the Reaction Device for Nucleic Acid Analysis) 
         [0053]    The microstructures formed on a substrate  601  used for the reaction device for nucleic acid analysis are not limited to the ones described above, and may be those explained below. Namely, other microstructures suitable for a substrate  601  used for the reaction device for nucleic acid analysis are explained with reference to  FIGS. 4 to 6 . 
         [0054]    The microstructures may be those composed of the first microstructures  602 , and the second microstructure  603 , the center point of which is inside of a square formed by the center points thereof. This second microstructure  603  is characterized to be lower in the height from the plane of the substrate  601  compared with the first microstructures  602 . Such the second microstructure  603  can be produced, for example, by using OEBR, ZEP2000, or the like, which is an analog-type resist, and by reducing the exposure dose in electron beam lithography of  603  compared with  602 . Alternatively, it may be produced by creating a stamper using a substrate having the first microstructures  602  as a master and using a nanoimprint lithography method as in  FIG. 3 . By immobilizing microparticles  604  while floating from the substrate using the substrate having two or more kinds of the microstructures  602 ,  603  with different heights like the above, it becomes possible to mitigate ghost imaging of intrinsic fluorescence from the substrate  601  during observation of the microparticles. Further, by providing space underneath the microparticles  604 , a matrix can be supplied uniformly to the surfaces of the microparticles  604 , which are fields of reaction, and enhancement of enzyme reaction efficiency becomes possible. By this, in the case of, for example, sequencing reactions shown in a nucleic acid analysis method described below, phase shifts can be prevented and reading of longer base lengths becomes possible. Also, by making the microparticles  604  float from the substrate surface, shortening of cleaning time can be attained due to enhancement of cleaning efficiency of the surfaces of the microparticles so that overall throughput is improved. Further, a phenomenon could occur that part of a reagent in a previous reaction cycle remains and reacts in a subsequent cycle in a reaction system such as shown in a nucleic acid analysis method described below, but by enhancing washability it becomes possible to reduce the amount of residue of the reagent by each cycle, yielding enhancement of reliability of data. 
         [0055]    Incidentally, in  FIG. 4 , the top drawing is a plan view showing the substrate  601  before the microparticle  604  is immobilized, the second drawing from the top is a cross-sectional view of the substrate  601  before the microparticle  604  is immobilized, and the bottom drawing is a cross-sectional view of the substrate  601  after the microparticle is arranged. With regard to this point, it is similar also in  FIG. 5  and  FIG. 6 . 
         [0056]    As structures for providing space underneath the microparticles  604  like the above, there are inverted pyramid structures  605  ( FIG. 5 ) or truncated pyramid structures  606  ( FIG. 6 ), or the like. These structures can be obtained by anisotropic etching of Si or the like. It should be noted that the inverted pyramid structures are produced by forming trenches on a substrate. 
         [0057]    (Regarding a Nucleic Acid Analyzer) 
         [0058]    Explanation is given on a nucleic acid analyzer of the present invention with reference to  FIG. 7 . A nucleic acid analyzer is provided with a means which supplies one or more kinds of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthetases, primers, and a nucleic acid sample, to a reaction device for nucleic acid analysis; a means which irradiates the reaction device for nucleic acid analysis with light; and a fluorescence detecting means which measures fluorescence of a fluorescent dye incorporated in a nucleic acid strand by a nucleic acid elongation reaction which occurs by co-presence of the nucleotides, the nucleic acid synthetases, and the nucleic acid sample on the reaction device for nucleic acid analysis. 
         [0059]    More specifically, a substrate  305  is installed to a reaction chamber composed of a cover plate  301  equipped with a channel in advance and an inlet  303  and an outlet  304  which are openings for exchange of solutions. Incidentally, as a material of the cover plate  301 , PDMS (polydimethylsiloxane) is used. After adjusting laser light  308  and  309  oscillating from an YAG laser light source (a wavelength of 532 nm and an output of 20 mW)  306  and an YAG laser light source (a wavelength of 355 nm and an output of 20 mW)  307  by a dichroic mirror  311  (which reflects light with a wavelength of 410 nm or shorter) so that the two of laser light are coaxial, they are concentrated by a lens  312 , and then are incident onto the substrate  305  via a prism  313  in a critical angle or greater. Fluorescence emitted from the cover plate  301  is converted to a collimated luminous flux by an objective lens  314 , background light and excitation light are blocked by an optical filter  315 , and an image is formed on a two-dimensional CCD camera  317  by an imaging lens  316 . 
         [0060]    Explanation is given on a variation example of the nucleic acid analyzer shown in  FIG. 7  with reference to  FIG. 8 . Differences from the nucleic acid analyzer shown in  FIG. 7  are installment of a temperature control element  419  under the reaction device and irradiation of laser light generated from light sources  406  and  407  via an optical fiber  418 . Because the reaction device is in direct contact with the temperature control element  419 , temperature difference between the temperature control element  419  and temperature of the solution on the reaction device can be made small, and efficiency of the sequencing reactions can be enhanced.  406  and  407  are laser light sources to radiate toward the reaction device from squares (opening parts) at their tips via the optical fiber  418 . 
         [0061]    (Regarding a Nucleic Acid Analysis Method Using the Reaction Device for Nucleic Acid Analysis and the Nucleic Acid Analyzer) 
         [0062]    In accordance with a method disclosed in Non-Patent Literature 2, after fragmentation and amplification of a DNA of a measurement object, beads on which the DNA fragments of measurement objects are immobilized are produced using the emulsion PCR method. Subsequently, in accordance with a method disclosed in Non-Patent Literature 6, the beads are immobilized on the reaction device for nucleic acid analysis of the present invention using acrylic gel. Next, the reaction device for nucleic acid analysis on which the beads are immobilized is installed to the nucleic acid analyzer shown in  FIG. 8 . Finally, in accordance with a method disclosed in the Non-Patent Literature 2, the following actions are performed to determine base sequences: 
         [0063]    (1) hybridization of anchor primers, 
         [0064]    (2) ligation of fluorescent primers, 
         [0065]    (3) detection of fluorescence, 
         [0066]    (4) removal of the anchor primers and the fluorescent primers, and 
         [0067]    (5) repeat of (1) through (4). 
         [0068]    Also, the present invention can use the successive reaction scheme other than the method by the above ligation. As nucleotides with fluorescent dyes, the one with a 3′-O-allyl group introduced as a protecting group at the 3′ OH position of ribose and with fluorescent dyes via allyl groups bound at the 5-position of pyrimidines or at the 7-position of purines can be used as disclosed in Non-Patent Literature 4. Since the allyl groups are cleaved by light irradiation or contact with palladium, quenching of the dyes and control of the elongation reactions can be attained simultaneously. Even in the successive reaction, removal of unreacted nucleotides by cleaning is not necessary. 
         [0069]    Further, in the present embodiment, since a cleaning step as shown in Non-Patent Literature 5 is not required, elongation reactions can also be measured in real time. As described above, by constructing the nucleic acid analyzer using the reaction device for nucleic acid analysis of the present embodiment, shortening of analysis time and simplification of the reaction device and the analyzer can be plotted without introducing a cleaning step, the elongation reactions of bases can also be measured not only in the successive reaction scheme but also in real time, and significant improvement of the throughput with respect to conventional technology can be designed. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           101 ,  201 ,  305 ,  501 ,  601  substrate 
           102 ,  503  microstructures 
           103 ,  604  microparticles 
           104  solution 
           202  regions where the microparticles are arranged in a lattice form 
           203  channel 
           204  reaction chamber 
           205  temperature control unit 
           206  dispensing unit 
           207  valve 
           208  waste liquid tank 
           301  cover plate 
           303  inlet 
           304  outlet 
           306 ,  307  YAG laser light source 
           308 ,  309  laser light 
           310  dichroic mirror 
           312  lens 
           313  prism 
           314  objective lens 
           315  optical filter 
           316  imaging lens 
           317  two-dimensional CCD camera 
           418  optical fiber 
           419  temperature control element 
           502  stamper 
           504  marker 
           602  first microstructures 
           603  second microstructures 
           605  inverted pyramid structures (microstructures) 
           606  square pole structures (microstructures)