JP2007256001A - Organic compound synthesizing apparatus and substrate for organic compound synthesis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an organic compound synthesizing apparatus and a substrate for organic compound synthesis capable of coping easily with a pattern change, and having a chemical substance in a spot having excellent uniformity. <P>SOLUTION: This organic compound synthesizer 100 is equipped with the substrate 110 for organic compound synthesis for holding an organic compound synthesized by a polymerization reaction of a repeated unit wherein a terminal is protected by a protection group, and a photoreactive material for generating a material for eliminating the protection group by a photoreaction; and a light irradiation device 130 having one or two or more light sources 132, for irradiating light toward the photoreactive material held on the substrate 110 for organic compound synthesis. The organic compound synthesizing apparatus 100 synthesizes one or two or more kinds of organic compounds on the substrate 110 for organic compound synthesis by allowing the light irradiation device 130 to move relatively to the substrate 110 for organic compound synthesis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an organic compound synthesizer and an organic compound synthesizer substrate, and more particularly to an organic compound synthesizer and an organic compound synthesizer substrate used for manufacturing a DNA chip in which DNAs having a desired base sequence are arranged in an array. About.

  In recent years, in the diagnosis of diseases using DNA (deoxyribonucleic acid), which is a biological material, a method using a chip in which DNA is arranged on a substrate is indispensable for realizing tailor-made medical care corresponding to individual differences. It is being studied and realized.

  Such a DNA chip has DNA segments with different arrangements arranged in a spot on a substrate. There are two methods for preparing DNA chips: a method of synthesizing DNA for each spot type in advance and arranging them on a substrate by an ink jet method, and a method of synthesizing by connecting nucleic acids on the substrate. Is considered.

  The latter method of synthesizing nucleic acids on a substrate is excellent in that DNA having an arbitrary sequence can be directly produced on a substrate using only materials corresponding to four bases. In producing such a DNA chip, for example, Affymetrix Corporation forms a DNA pattern on a substrate by a photolithography method. Further, for example, in Agilent, the basic method is the same as that of Affymetrix, but DNA is patterned by an inkjet method (see, for example, Patent Documents 1 to 4).

  In general, the amidite method is often used as a specific method for producing the DNA chip as described above. The amidite method will be briefly described below. First, a protecting group is formed on a substrate via a linker. This protecting group is removed by reaction to light or acid, and the end of the linker with hydrogen appears. When a base modified with a protecting group is reacted here, the part having the isopropyl group of the base is connected to the linker. Further, when a light is irradiated or an acid is supplied to a place where a new base is to be connected, the protecting group is removed, and when a base modified with the protecting group is reacted therewith, the new base is connected to that part. By repeating such a reaction, a necessary base sequence can be formed.

US Pat. No. 6,372,483 US Pat. No. 6,420,180 US Pat. No. 6,600,031 US Pat. No. 6,375,903

  By the way, in what place on the substrate the DNA of which base sequence is formed can be determined by determining the pattern of the position where the light is irradiated or the position where the acid is spotted (supplied). For such a pattern on a substrate such as light irradiation, a glass mask used at the time of manufacturing a semiconductor integrated circuit (IC) is mainly used at present. This method using a glass mask is suitable for mass production of the same pattern, but it is difficult to respond immediately to the pattern change.

  As a method corresponding to such a pattern change, for example, a method using a micromachine mirror developed for a display or a liquid crystal has been devised. However, when such a micromachine mirror or liquid crystal is used, there is a problem that the uniformity of chemical substances such as DNA in the spot is inferior to the method using a glass mask. Furthermore, as with the method using a glass mask, the use of a projection optical system has limited the downsizing of the apparatus.

  As another method corresponding to the pattern change, there is a method of spotting by an ink jet method. However, the inkjet method has a problem in that the uniformity of each DNA section is inferior due to the fact that the position alignment for each spotting is required to be precise and unevenness occurs during drying.

  Therefore, the present invention has been made in view of such problems, and the object thereof is a new and improved organic material that can easily cope with pattern change and has excellent uniformity of chemical substances in the spot. The object is to provide a compound synthesis apparatus and a substrate for organic compound synthesis.

  In order to solve the above problems, according to one aspect of the present invention, there is provided an organic compound synthesizer for synthesizing an organic compound composed of one or more repeating units by light irradiation, and the terminal is protected with a protecting group. A repeating unit and a photoreactive substance that generates a photogenerating substance that removes a protecting group by a photoreaction and is supplied with a reaction container in which a polymerization reaction of the repeating unit is performed, and one or more light sources. An organic compound synthesizer is provided that includes a light irradiation device that irradiates light into the reaction vessel.

  The organic compound synthesizer may be configured to synthesize one or more organic compounds by moving the light irradiation device relative to the reaction vessel.

  Further, the light irradiation device may be configured to selectively irradiate a part of the photoreactive substances among the photoreactive substances supplied into the reaction container when moving relative to the reaction container. Good.

  In the organic compound synthesizer, the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance may be performed at substantially the same position.

  On the other hand, in the organic compound synthesizer, the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance may be performed at separate positions. When the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance are performed at substantially the same position, the light irradiated from the light irradiation device may be directly irradiated to the synthesized organic compound, which may have an adverse effect. However, it is possible to improve the reaction efficiency of the polymerization reaction without adversely affecting the synthesized organic compound by configuring the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance to be separated from each other. Can do.

  In the organic compound synthesizer, the light irradiation device may have two or more light sources arranged in an array, and the light irradiation device is arranged in a direction perpendicular to the axial direction of the light source array. You may comprise so that it may move relatively.

  Moreover, as a light source of a light irradiation apparatus, a light emitting diode, a semiconductor laser, etc. can be used, for example, It is preferable to use a light emitting diode especially.

  Furthermore, the light irradiation device may further include one or more condensing lenses that condense light emitted from the light source.

  In addition, for example, as the photoreactive substance, a proton supply substance that releases protons by a photoreaction can be used, and as the protective group, a functional group that is released from the repeating unit by reacting with a proton is used. can do. In addition, as the photoreactive substance, for example, a substance that releases oxygen radicals may be used.

  In order to solve the above problems, according to another aspect of the present invention, there is provided an organic compound synthesis substrate on which an organic compound synthesized using the organic compound synthesis apparatus described above is fixed.

  In addition, the organic compound synthesis substrate may have, for example, a plurality of photoreactive substance holding units for holding photoreactive substances. In this case, the polymerization reaction of the repeating unit is performed in a photoreactive substance holding unit that holds a photoreactive substance irradiated with light.

  In addition to the above examples, the organic compound synthesizing substrate may have a structure that can transmit, for example, repeating units and protons released from the photogenerating substance. In this case, the photoreaction of the photoreactive substance is performed on the surface of the organic compound synthesis substrate on the side irradiated with light, and the polymerization reaction of the repeating unit is opposite to the side of the organic compound synthesis substrate irradiated with light. It is performed at the position where the photoreaction of the photoreactive substance is performed on the side surface.

  Further, in addition to the above example, for example, the organic compound synthesis substrate may have a plurality of through holes penetrating the substrate, and the through hole has an opening on the side irradiated with light. The opening on the side opposite to the side irradiated with light may be closed by a reactive substance permeable member capable of transmitting the repeating unit and the photogenerating substance released from the photoreactive substance. In this case, the photoreaction of the photoreactive substance is performed in the vicinity of the opening on the side irradiated with light, and the polymerization reaction of the repeating unit is irradiated with light from the through-hole in which the photoreaction of the photoreactive substance has been performed. This is performed on the surface of the reactant permeable member that closes the opening on the side opposite to the open side.

  In addition, for example, as the photoreactive substance, a proton supply substance that releases protons by a photoreaction can be used, and as the protective group, a functional group that is released from the repeating unit by reacting with a proton is used. can do. In addition, as the photoreactive substance, for example, a substance that releases oxygen radicals may be used.

  According to the present invention, it is possible to provide an organic compound synthesizing apparatus and an organic compound synthesizing substrate that can easily cope with a pattern change and have excellent uniformity of chemical substances in a spot.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the present specification and drawings, components having substantially the same functional configuration are denoted by the same reference numerals, and redundant description is omitted.

  As described above, the organic compound synthesis apparatus according to the present invention is an apparatus that synthesizes an organic compound composed of one or more repeating units by light irradiation. Examples of organic compounds synthesized by this apparatus include biopolymer compounds such as nucleic acids (DNA, RNA, etc.) or proteins, and ordinary polymer compounds (polymers, oligomers) other than living organisms. In the case of nucleic acids such as DNA and RNA, 4 types of bases (A, T, G, C) are repeating units. In the case of proteins, various amino acids are repeating units. In this case, a monomer is a repeating unit.

  Hereinafter, as an organic compound synthesizer according to the present invention, a DNA array synthesizer for synthesizing DNA arranged in an array will be described as an example.

(First embodiment)

  The organic compound synthesizer according to the first embodiment of the present invention described below is an example in which the above-described polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance are performed at substantially the same position.

<Configuration of DNA Array Synthesizer 100>
First, the configuration of a DNA array synthesizer 100 as an example of an organic compound synthesizer according to the first embodiment of the present invention will be described with reference to FIG. FIG. 1 is a cross-sectional view showing the overall configuration of the DNA array synthesizer 100 according to this embodiment.

  As shown in FIG. 1, a DNA array synthesizer 100 according to this embodiment includes a DNA array substrate 110, a reaction vessel 120, and a light irradiation device 130 as an example of an organic compound synthesis substrate according to this embodiment. Prepare.

  The DNA array substrate 110 is a light that generates DNA that is synthesized by a polymerization reaction of four types of bases (A, T, G, C) whose ends are protected by a protecting group, and a substance that releases the protecting group by a photoreaction. A substrate for holding the reactant 30.

  In the present embodiment, the protecting group for protecting the terminals of the four types of bases is a functional group that is released from the terminal of the base by reacting with a proton in the acidic region, for example, 5′-dimethoxy. For example, a 5'-dimethyltril or the like can be used.

  In addition, as the photoreactive substance 30 according to the present embodiment, for example, a proton supply substance that releases protons by a photoreaction or a photogenerating substance such as an oxygen radical can be used. As such a proton supply substance (PGA), for example, triaryl-sulfonium-hexafluoroantimonate dissolved in polypropylene carbonate can be used. In this case, protons can be released by irradiating the PGA with light having a wavelength of about 365 nm. In addition, as PGA, a photoacid generator (PAG) for visible light or a combination of a photosensitizer and a photoacid generator can be used.

  Examples of the visible light photoacid generator include nonafluorobutanesulfonic acid 1,3,6-trioxo-3.6-dihydro-1H-11-thia-azacyclopenta [a] anthracen-2-yl ester. And nonafluorobutanesulfonic acid 8-isopropyl-1,3,6-trioxo-3.6-dihydro-1H-11-thia-azacyclopenta [a] anthracen-2-yl ester. In this case, protons can be released by irradiating the PGA with light having a wavelength of about 436 nm.

  On the other hand, examples of the acid generator include iodonium salts such as onium salt and diallyl iodonium, and sulfonium salts such as triallylsulfonium and dialkylphenacylsulfonium. Examples of the photosensitizer include, for example, thioxanthone such as 2-isopropylthioxanthone and 1-chloro-4-propoxythioxanthone, ketocoumarin, phenothiazine derivative (Phenothiazine), camphorquinone, and benzene. Derivatives (Benzil), titanocene compounds (Titanocene), compounds having a QBS benzosulfanyl group, and the like. For the photosensitizer, for example, light having a wavelength of about 383 nm for thioxanthone, light having a wavelength of about 478 nm for camphorquinone, and wavelength of about 480 to 486 nm for a benzene derivative. By irradiating light and a titanocene compound with light having a wavelength of about 460 nm, protons can be released in combination with an acid generator.

  Moreover, as a material of the DNA array substrate 110, glass, various plastics, or the like can be used.

  Here, the configuration of the DNA array substrate 110 will be described with reference to FIG. 2A and 2B are diagrams showing the configuration of the DNA array substrate 110 according to this embodiment, wherein FIG. 2A is a plan view and FIG. 2B is a partial cross-sectional view.

  As shown in FIG. 2, the DNA array substrate 110 has a concave portion 112 as an example of a photoreactive substance holding part for holding a photoreactive substance 30 (hereinafter also referred to as “proton supply substance 30”). A plurality are formed. The reason why such a photoreactive substance holding portion such as the recess 112 is provided is because it is necessary to localize a chemical reactive substance such as proton generated by the photoreaction of the photoreactive substance 30 within a predetermined region. That is, if a chemical reaction substance such as proton is not localized in a predetermined region, interference between adjacent sections among sections where each DNA is synthesized occurs, and a target product (in this embodiment, This is because something other than (DNA) may be produced.

  Various methods such as surface modification of the DNA array substrate 110 are conceivable as a method for localizing the chemical reaction material. In this embodiment, a plurality of recesses 112 are provided on the DNA array substrate 110. Then, the focal point of the light emitted from the light irradiation device 130 to be described later is connected in the recess 112, so that the photochemical reaction of the photoreactive substance 30 occurs only in the desired recess 112 irradiated with light, and protons. A chemical reaction material such as is localized in the desired recess 112.

  The reaction vessel 120 is an embodiment of a reactant supply unit that supplies a repeating unit whose end is protected with a protecting group and a photoreactive material to an organic compound synthesis substrate. That is, the reaction vessel 120 supplies the DNA array substrate 110 with a reaction solution containing a desired base and the proton supply material 30 among the four types of bases whose ends are protected with a protecting group.

  This reaction container 120 has a predetermined space (the following reaction) for holding the reaction solution so as to cover one surface of the DNA array substrate 110 (in this embodiment, the other surface of the light irradiation device 130). The reaction liquid inlet 122, the reaction liquid outlet 124, and the reaction liquid holder 126 are mainly provided. The reaction liquid holding unit 126 holds the reaction liquid during the DNA extension reaction (polymerization reaction). The reaction solution is introduced from the reaction solution inlet 122, passes through the reaction solution holding unit 126, and is discharged from the reaction solution outlet 124. As described above, in the reaction vessel 120, a reaction liquid flow path is formed as the reaction liquid inlet 122 → the reaction liquid holding unit 126 → the reaction liquid outlet 124. As a specific structure of the reaction vessel 120, for example, a so-called microfluidic structure formed by digging a groove on a substrate is typically used.

  The light irradiation device 130 has a plurality of light sources 132 and irradiates light from the light sources 132 toward the proton supply material 30 held on the DNA array substrate 110. Here, in the present embodiment, a plurality of light sources 132 are provided, but the light irradiation device according to the present invention may have a configuration in which only one light source is provided. Thus, by irradiating the proton supply material 30 with light from the light source 132 of the light irradiation device 130, the chemical reaction system constituted by the DNA array substrate 110 and the reaction vessel 120 has a spatial distribution. Photochemical reaction can be caused.

  Here, the configuration of the light irradiation device 130 will be described in detail with reference to FIG. FIG. 3 is a perspective view showing the configuration of the light irradiation device 130 according to the present embodiment.

  As shown in FIG. 3, the light irradiation device 130 mainly includes a plurality of light sources 132 arranged in an array on a light source array substrate 134 and a rod lens array 138 in which rod lenses 136 are arranged in an array. Prepare.

  Although the plurality of light sources 132 according to the present embodiment are arranged in one row in an array, they are not necessarily arranged in an array, and the number of light sources 132 is not limited to one row. The size of the DNA array substrate 110, the light irradiation pattern formed on the DNA array substrate 110, and the like can be changed as appropriate. Further, the number of the light sources 132 can be appropriately selected depending on the size of the DNA array substrate 110, the light irradiation pattern formed on the DNA array substrate 110, and the like.

  As the light source 132, for example, an optical element using a semiconductor can be used. As such an optical element, for example, a light emitting diode or a semiconductor laser can be used. In selecting the emission wavelength, it is possible to select a desired emission wavelength according to the light energy necessary for synthesizing an organic compound such as DNA. It is preferable to use a light source that emits short light. On the other hand, light emitting diodes and semiconductor lasers are easier to produce as they emit light at longer wavelengths. From this point of view, in this embodiment, for example, a blue light emitting diode having an emission wavelength of about 400 nm can be used as the light source 132.

  When a light emitting diode is used as the light source 132, the pitch of the light emitting diodes (interval between adjacent light emitting diodes) can be about 20 μm, and the focal length of the rod lens 136 can be about 4 mm. Furthermore, by using a light emitting diode as the light source 132, it is possible to obtain a brightness about 100 to 1000 times higher than that of a conventionally used UV lamp or the like, so that the reaction time of the photoreactive substance 30 can be shortened. .

  Since the focal length can be shortened by using the light emitting diode array as compared with the case of using a conventional UV lamp or laser, the organic compound synthesizing substrate such as the DNA array substrate 110 and the light irradiation device 130 The distance can be shortened, whereby the size of the entire organic compound synthesizer such as the DNA array synthesizer 100 can be reduced.

  The rod lens 136 is an example of a condensing lens according to the present embodiment that condenses the light emitted from the light source 132 and adjusts the light to a desired focal position. In the present embodiment, as described above, in order to cause the photochemical reaction (proton release) of the proton supply substance 30 in the recess 112, the focal position of the light irradiated from the light source 132 is in the recess 112. , And are adjusted by the rod lens 136.

  In the present embodiment, the rod lenses 136 are arranged in an array so as to form a staggered arrangement. Then, the light from the plurality of light sources 132 arranged in an array is condensed to adjust the focal position. However, as in the case of the light source 132, the rod lenses 136 are not necessarily arranged in an array, and the arrangement of the rod lenses 136 is not limited. Further, the number of rod lenses 136 is not particularly limited.

  In this embodiment, the light irradiation device 130 is disposed on the opposite side of the DNA array substrate 110 from the reaction vessel 120 (that is, the opposite side to the side where DNA synthesis is performed), and emits light toward the DNA array substrate 110. It is configured to irradiate. This prevents light from being absorbed in the reaction liquid holding unit 126, and the amount of irradiated light is reduced on the DNA array substrate 110, thereby preventing the efficiency of the photoreaction of the photoreactive substance 30 from being reduced. Because. However, when the thickness of the reaction solution holding unit 126 (in the direction perpendicular to the DNA array substrate 110) is thin and the decrease in the amount of light on the DNA array substrate 110 can be suppressed, the same side of the DNA array substrate 110 as the reaction container 120 is used. There is no problem even if it is irradiated with light.

  Further, the DNA array synthesizer 100 according to the present embodiment has a moving mechanism (not shown) that moves the light irradiation device 130 relative to the DNA array substrate 110. For example, the moving mechanism can be configured to horizontally move the light irradiation device 130 parallel to the DNA array substrate 110 in a direction perpendicular to the axial direction of the array of the light sources 132. However, the direction of relative movement of the light irradiation device 130 is not limited to the above case, and may be, for example, the diagonal direction of the DNA array substrate 110, or the light irradiation device 130 may be along the side direction of the DNA array substrate 110. It may be configured to move relatively while reciprocating.

  As described above, the light irradiation device 130 is moved relative to the DNA array substrate 110 to turn on and off the light irradiation of the light source 134 temporally and spatially, and a desired position at which the photochemical reaction of the photoreactive substance 30 is caused to occur. By selectively irradiating with light, a spatial pattern of a position where a photochemical reaction occurs (a position where light is irradiated by the light source 134 among the plurality of recesses 112) can be formed on the DNA array substrate 110. Further, as described above, the focal position of the light irradiated from the light source 132 of the light irradiation device 130 is adjusted by the rod lens 136 in the desired concave portion 112 where the photochemical reaction is desired to occur. A reaction will occur.

  When it is desired to change the spatial pattern of light irradiation (the spatial pattern of the position where photochemical reaction occurs) by forming a spatial pattern (pattern of light irradiation) where the photochemical reaction occurs by such a method, a light irradiation device When the 130 is relatively moved, it is possible to easily cope with the pattern change only by controlling the on / off timing of light irradiation from the light source 132.

  Furthermore, by supplying a desired base to which DNA extension is to be performed among the four types of bases that are repeating units according to the present embodiment, to the DNA array substrate 110 on which such a spatial pattern is formed, In the concave portion 112 (photoreactive substance holding portion) that has been irradiated with light, DNA is elongated (polymerization reaction of repeating units). By repeating such an operation, a DNA array in which one or more kinds of DNAs having a desired base sequence are arranged in an array can be produced. Details of the method for manufacturing such a DNA array will be described later.

  Here, in the present embodiment, the light irradiation device 130 having a structure in which the light source 132 and the rod lens 136 are arranged in an array is used, and the light irradiation device 130 is placed in a direction perpendicular to the axis of the array in the DNA array substrate. A spatial pattern of positions where photochemical reaction occurs is formed on the DNA array substrate 110 by being moved relative to 110. By forming the spatial pattern in this manner, the light irradiation device 130 is simply moved relative to the DNA array substrate 110, and the chemical substance such as DNA in the spot is irradiated with light accurately at a desired position. Uniformity can be improved and a spatial pattern can be formed at high speed.

  The configuration of the DNA array synthesizer 100 according to this embodiment has been described above. Next, a DNA array synthesis method using the DNA array synthesizer 100 will be described.

<Method of synthesizing DNA array using DNA array synthesizer 100>
Hereinafter, a DNA array synthesis method using the DNA array synthesizer 100 according to the present embodiment will be described in detail with reference to FIG. 4A and 4B are explanatory diagrams showing a method for synthesizing a DNA array according to the present embodiment, in which FIG. 4A shows a state before a DNA elongation reaction, FIG. 4B shows a state in which a protecting group has been removed, and FIG. ) Shows the state during the DNA extension reaction, and (d) shows the state after the DNA extension reaction.

  First, as shown in FIG. 4 (a), a first base 26 (in this example, cytosine (T)) having a protecting group 24 is formed in the recess 112 of the DNA array substrate 110 via a linker 22. Here, a reaction liquid 12 containing a proton supply substance (not shown in FIG. 4A) that emits protons by light is allowed to flow using a reaction liquid supply unit (not shown in FIG. 4A). .

  Next, as shown in FIG. 4B, when the light irradiated from the light irradiation device 130 is collected in a place where a DNA elongation reaction is to occur, for example, the concave portion 112 on the left side of FIG. The proton supply substance present in the left concave portion 112 causes a photoreaction to supply protons, and an acidic region 40 is generated in the vicinity of the focal position of the light irradiated from the light irradiation device 130. Then, when the protecting group 24 of the base 26 existing in the acidic region 40 reacts with protons, the protecting group 24 is removed from the base 26 to generate a base 26 that can react with other bases.

  Next, as shown in FIG. 4C, a second base 28 (this example) to be reacted with the reactable base 26 using a reaction liquid supply unit (not shown in FIG. 4C). Then, when the amidite solution 14 containing adenine (A) is flowed, the second base 28 having a protecting group is bonded to the first base 26 from which the protecting group 24 has been released, and DNA elongation occurs. On the other hand, since the first base 26 held in the section not irradiated with light, for example, the concave portion 112 on the right side of FIG. 4C, has the protecting group 24, the second base 28 may be bound thereto. It cannot be done and DNA elongation does not occur.

  In this way, as shown in FIG. 4D, a new base (in this example, the second base in the left recess 112 in this example) is added only to the desired DNA (in this example, the second base 26). Can be caused to cause a DNA elongation reaction selectively.

  Further, after the reaction system including the DNA array substrate 110 is washed with the washing liquid, the cycle shown in FIGS. 4A to 4D is repeated again, so that a desired position on the DNA array substrate 110 is obtained. DNA having a desired sequence and length can be formed. In this case, if a plurality of recesses 112 are arranged in an array on the DNA array substrate 110, a DNA array in which a plurality of types of DNAs having desired sequences and lengths are arranged in an array can be manufactured. it can.

  However, in the above-described first embodiment, the position where protons are generated in order to remove the protecting group, that is, the position where light is irradiated by the light irradiation device and the position where the DNA elongation reaction is performed are the same recess. 112. Thus, when the light irradiation position is the same as the position where the DNA elongation reaction occurs, the light irradiated from the light irradiation apparatus is directly irradiated to the DNA held on the DNA array substrate, It will have an adverse effect. As such an adverse effect, for example, there is a possibility that DNA may be mutated by intense light irradiated from a light irradiation device or heat generated by a photoreaction of a proton-emitting substance.

  Therefore, in the second embodiment described below, by separating the position where the proton is generated by irradiating light and the position where the DNA elongation reaction is performed without adversely affecting the synthesized DNA, The reaction efficiency of the DNA synthesis reaction is improved.

(Second Embodiment)

  The organic compound synthesizer according to the second embodiment of the present invention to be described below is an example of an apparatus in which the above-described polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance are performed at a separated position.

<Configuration of DNA Array Synthesizer 200>
Hereinafter, the configuration of a DNA array synthesizer 200 as an example of an organic compound synthesizer according to the second embodiment of the present invention will be described with reference to FIG. FIG. 5 is a cross-sectional view showing the overall configuration of the DNA array synthesizer 200 according to this embodiment.

  As shown in FIG. 5, a DNA array synthesizer 200 according to this embodiment includes a DNA array substrate 210, a reaction vessel 220, and a light irradiation device 230 as an example of an organic compound synthesis substrate according to this embodiment. Prepare.

  The DNA array substrate 210 is a light that generates DNA that is synthesized by a polymerization reaction of four types of bases (A, T, G, C) whose ends are protected by a protecting group, and a substance that releases the protecting group by a photoreaction. A substrate for holding the reactant 30.

  Since the protecting group and the photoreactive substance 30 in this embodiment are the same as those in the first embodiment described above, detailed description thereof is omitted.

  Moreover, as a material of the DNA array substrate 210, glass, various plastics, or the like can be used.

  Here, the configuration of the DNA array substrate 210 will be described with reference to FIG. FIG. 6 is a cross-sectional view showing the configuration of the DNA array substrate 210 according to the present embodiment. (A) is a first example, (b) is a second example, and (c) is a third example. Indicates.

  In the DNA array substrate 210 according to the present embodiment, at least a DNA section where individual DNA is held or a peripheral portion thereof from one surface of the DNA array substrate 210 (the surface on the light irradiation device 230 side in FIG. 6). A flow channel structure is provided in which the reaction liquid flows to the other surface (the surface on the other side of the light irradiation device 230 in FIG. 6).

  As a first example of such a flow channel structure, as shown in FIG. 6A, a DNA array substrate 210 is allowed to pass through a reaction solution containing a base as a repeating unit and protons released from the proton supply material 30. A configuration having a possible structure is mentioned. For example, a porous member 212 such as a porous glass substrate or a plastic film can be used as a structure that can permeate the reaction solution. In the first example, as described in the first embodiment, it is necessary to localize a chemical reaction substance such as a proton generated by the photoreaction of the photoreaction substance 30 in a predetermined region. For this reason, it is desirable to modify the surface of the DNA array substrate 210 or provide a recess on the surface of the DNA array substrate 210.

  As a second example, as shown in FIG. 6B, the DNA array substrate 210 is provided with a plurality of through holes 210a penetrating the substrate, and one opening portion of the through hole 210a (this embodiment) In the embodiment, the opening on the light irradiation device 230 side) is opened, and the other opening (in this embodiment, the opening on the other side of the light irradiation device 230) is the base and the proton released from the proton supply substance 30. The structure which is obstruct | occluded by the reactive substance permeation | transmission member which can permeate | transmit the reaction liquid containing this is mentioned. As the reactant permeable material, the porous member 212 mentioned in the first example can be used. In this second example, the porous member 212 is disposed only in the other opening of the through hole 210a. However, the DNA array includes not only the opening but also the opening of the through hole 210a. You may comprise so that a part of surface of the board | substrate 210 may be covered.

  In addition, as shown in FIG. 6C, the third example is the same as the second example in that a through-hole 210a is provided in the DNA array substrate 210, while the other opening portion of the through-hole 210a ( In the present embodiment, in order to close the opening on the other side of the light irradiation device 230, the reactive substance transmitting member such as the porous member 212 covers the entire surface, not a part of the surface of the DNA array substrate 210. This is different from the second example in that they are arranged as described above.

  The reaction vessel 220 is an embodiment of a reactant supply unit that supplies a repeating unit whose end is protected with a protecting group and a photoreactive material to an organic compound synthesis substrate. That is, the reaction vessel 220 supplies a reaction solution containing a desired base and the proton supply substance 30 among the four types of bases whose ends are protected with a protecting group to the DNA array substrate 210.

  The reaction vessel 220 is a predetermined space for holding the reaction solution so as to cover both surfaces of the DNA array substrate 210 (see the inlet side reaction solution holding unit 226A and the outlet side reaction solution holding unit 226B below). Are disposed on both sides of the DNA array substrate 210. The reaction vessel 220 mainly includes a reaction liquid inlet 222, a reaction liquid outlet 224, an inlet side reaction liquid holding part 226A, and an outlet side reaction liquid holding part 226B.

  For example, the reaction liquid inlet 222 is disposed on the light irradiation device 230 side with respect to the DNA array substrate 210, and the reaction liquid outlet 224 is, for example, on the opposite side of the reaction liquid inlet 222, that is, on the DNA array substrate 210. On the other hand, it is disposed on the other side of the light irradiation device 230. Further, the reaction solution holding unit 226A and the outlet side reaction solution holding unit 226B hold the reaction solution during the DNA extension reaction (polymerization reaction). The reaction solution is introduced from the reaction solution inlet 222, passes through the inlet-side reaction solution holding unit 226A, passes through the porous member 212, or passes through the through-hole 210a of the DNA array substrate 210, and then exits from the reaction solution. It passes through the holding part 226B and is discharged from the reaction solution outlet 224. Thus, in the reaction vessel 220, a reaction liquid flow path is formed in the order of the reaction liquid inlet 222 → the inlet side reaction liquid holding part 226 A → the outlet side reaction liquid holding part 226 B → the reaction liquid outlet 224.

  The light irradiation device 230 includes a plurality of light sources 232 and a plurality of rod lenses 236 as an example of a condensing lens that collects light emitted from the light sources 232, and the light source 232 passes through the rod lenses 236. Light is irradiated toward the proton supply material 30 held on the DNA array substrate 210. Here, in the present embodiment, a plurality of light sources 232 are provided, but the light irradiation device according to the present invention may have a configuration in which only one light source is provided. In this way, by irradiating the proton supply material 30 with light from the light source 232 of the light irradiation device 230, the chemical reaction system constituted by the DNA array substrate 210 and the reaction vessel 220 has a spatial distribution. Photochemical reaction can be caused.

  Although not shown, the plurality of light sources 232 according to the present embodiment are arranged in an array on a light source array substrate (not shown), and the plurality of rod lenses 236 are A rod lens array (arrayed in one row) is arranged in a row so that one rod lens 236 is arranged for one light source 232 at a position corresponding to each of the plurality of light sources 232 arranged in an array. (Not shown).

  As described above, the plurality of light sources 232 and the plurality of rod lenses 236 according to the present embodiment are arranged in one row in an array, but are not necessarily arranged in an array. The number of arrangement of the rod lenses 236 is not limited to one, and can be appropriately changed depending on the size of the DNA array substrate 210, the pattern of light irradiation formed on the DNA array substrate 210, and the like. Further, the number of the light sources 232 and the rod lenses 236 can be appropriately selected according to the size of the DNA array substrate 210, the light irradiation pattern formed on the DNA array substrate 210, and the like.

  As the light source 232, for example, an optical element using a semiconductor can be used. As such an optical element, for example, a light emitting diode or a semiconductor laser can be used. In selecting the emission wavelength, it is possible to select a desired emission wavelength according to the light energy necessary for synthesizing an organic compound such as DNA. It is preferable to use a light source that emits short light. On the other hand, light emitting diodes and semiconductor lasers are easier to produce as they emit light at longer wavelengths. From this point of view, in the present embodiment, for example, a blue light emitting diode having an emission wavelength of about 400 nm can be used as the light source 232.

  When a light emitting diode is used as the light source 232, the pitch of the light emitting diodes (interval between adjacent light emitting diodes) can be about 20 μm, and the focal length of the rod lens 236 can be about 4 mm. Furthermore, by using a light emitting diode as the light source 232, it is possible to obtain a brightness about 100 to 1000 times higher than that of a conventionally used UV lamp or the like, so that the reaction time of the photoreactive substance 30 can be shortened. .

  Since the focal length can be shortened by using the light emitting diode array as compared with the case of using a conventional UV lamp or laser, the organic compound synthesizing substrate such as the DNA array substrate 210 and the light irradiation device 230 The distance can be shortened, whereby the size of the entire organic compound synthesizer such as the DNA array synthesizer 200 can be reduced.

  Further, in this embodiment, when the photochemical reaction (proton release) of the proton supply substance 30 is performed using the DNA array substrate of the first example shown in FIG. 6A, the DNA array substrate is configured. In order to cause it to occur on the surface of the porous member 212 on the light irradiation device 230 side, the rod lens 236 causes the focal position of the light irradiated from the light source 232 to be the surface of the porous member 212 on the light irradiation device 230 side. Adjusted. When the DNA array substrates of the second and third examples shown in FIGS. 6B and 6C are used, the through-hole 210a formed in the DNA array substrate 210 on the light irradiation device 230 side. In order to make it raise in the vicinity of an opening part, it adjusts with the rod lens 236 so that the focus position of the light irradiated from the light source 232 may become near the opening part by the side of the light irradiation apparatus 230 of the through-hole 210a.

  Thus, protons are generated by the photochemical reaction of the proton supply material 30 at the light collection position adjusted by the rod lens 236 (the focal position of the light emitted from the light source 232). The generated protons reach the surface on the other side of the light irradiation device 230 of the DNA array substrate 210 by the diffusion of the protons themselves or the flow of the reaction solution.

  On the other hand, the DNA synthesis reaction is performed on the other surface of the light irradiation device 230 of the DNA array substrate 210. More specifically, in the case of the first example, the DNA synthesis reaction is performed on the other surface of the light irradiation device 230 of the porous member 212 constituting the DNA array substrate. In the case of the third example, it is performed on the surface of the porous member 212 that closes the opening on the other side of the light irradiation device 230 of the through hole 210a.

  As described above, in this embodiment, the position where the photoreaction is caused by light irradiation to generate protons and the position where the DNA synthesis reaction is performed are separated from each other. In contrast, the reaction efficiency of the DNA synthesis reaction can be improved without adversely affecting the synthesized DNA.

  In the case of the second example (see FIG. 6B) and the third example (see FIG. 6C), the other opening of the through hole 210a is blocked by the porous member 212. Although the configuration is shown, a configuration in which both openings of the through hole 210a are opened and the porous member 212 is not provided may be employed. In this case, the DNA synthesis reaction can be performed around the wall surface of the through hole 210 a or the opening of the through hole 210 a on the DNA array substrate 210.

  The DNA array synthesizer 200 according to this embodiment has a moving mechanism (not shown) that moves the light irradiation device 230 relative to the DNA array substrate 210. For example, the moving mechanism can be configured to horizontally move the light irradiation device 230 in parallel to the DNA array substrate 210 in a direction perpendicular to the axial direction of the array of the light sources 232. However, the direction of relative movement of the light irradiation device 230 is not limited to the above case, and may be, for example, the diagonal direction of the DNA array substrate 210, or the light irradiation device 230 extends along the side direction of the DNA array substrate 210. It may be configured to move relatively while reciprocating.

  In this way, the light irradiation device 230 is moved relative to the DNA array substrate 210 to turn on and off the light irradiation of the light source 234 temporally and spatially, and a desired position where the photochemical reaction of the photoreactive substance 30 is desired to occur. By selectively irradiating with light, a spatial pattern where the photochemical reaction occurs can be formed on the DNA array substrate 210. Further, as described above, the focus position of the light emitted from the light source 232 of the light irradiation device 230 is adjusted by the rod lens 236 to a desired position where the photochemical reaction is to occur. Will happen.

  When it is desired to change the spatial pattern of light irradiation (the spatial pattern of the position where photochemical reaction occurs) by forming a spatial pattern (pattern of light irradiation) where the photochemical reaction occurs by such a method, a light irradiation device When the 230 is relatively moved, it is possible to easily cope with the pattern change only by controlling the on / off timing of light irradiation from the light source 232.

  Furthermore, by supplying the DNA base substrate 210 on which such a spatial pattern is formed, among the four types of bases that are repeating units according to the present embodiment, a desired base to be subjected to DNA extension is supplied. DNA extension (polymerization reaction of repeating units) is performed at a position corresponding to the position where light irradiation has been performed. By repeating such an operation, a DNA array in which one or more kinds of DNAs having a desired base sequence are arranged in an array can be produced. Details of the method for manufacturing such a DNA array will be described later.

  Here, in the present embodiment, the light irradiation device 230 having a structure in which the light source 232 and the rod lens 236 are arranged in an array is used, and the light irradiation device 230 is placed in a direction perpendicular to the axis of the array. A spatial pattern of positions where photochemical reaction occurs is formed on the DNA array substrate 210 by being moved relative to 210. By forming the spatial pattern in this manner, the light irradiation device 230 is simply moved relative to the DNA array substrate 210, and the chemical substance such as DNA in the spot is irradiated with light accurately at a desired position. Uniformity can be improved and a spatial pattern can be formed at high speed.

  The configuration of the DNA array synthesizer 200 according to the present embodiment has been described above. Next, a DNA array synthesis method using the DNA array synthesizer 200 will be described.

<Method of synthesizing DNA array using DNA array synthesizer 200>
Hereinafter, a DNA array synthesis method using the DNA array synthesizer 200 according to the present embodiment will be described in detail with reference to FIG. FIG. 7 is an explanatory diagram showing a method for synthesizing a DNA array according to the present embodiment. (A) is a state before a DNA extension reaction, and (b) is an acidic region formed by a photoreaction. The state, (c) shows the state where the acidic region has moved and the protecting group has been removed, (c) shows the state during the DNA extension reaction, and (d) shows the state after the DNA extension reaction.

  In the following description, for convenience of explanation, a case where the substrate according to the second example shown in FIG. 6B is used as the DNA array substrate 210 will be described as an example. The same applies to.

  First, as shown in FIG. 7 (a), a first base 26 having a protecting group 24 via a linker 22 on a porous member 212 that closes one opening of a through hole 210a of a DNA array substrate 210 (this In the example, cytosine (T)) is formed. Next, a reaction liquid 12 containing a proton supply substance (omitted in FIG. 7A) that releases protons by light using a reaction liquid supply unit (not shown in FIG. 7A) is used. Shed.

  Next, as shown in FIG. 7B, the light irradiated from the light irradiation device 230 is applied to a place where a photochemical reaction is to occur, for example, in the vicinity of the opening of the left through-hole 210a in FIG. When the light is condensed, the proton supply substance existing in the vicinity of the opening of the left through-hole 210a undergoes a photoreaction to supply protons, and the acidic region 40 is located in the vicinity of the focal position of the light emitted from the light irradiation device 230. Produces. At this stage, the acidic region 40 is separated from the position where the DNA synthesis reaction is performed.

  Further, as shown in FIG. 7C, the acidic region 40 is caused to pass through the through-hole 210a due to diffusion of protons generated in the flow path of the reaction solution formed in the reaction vessel 220 described above or the flow of the reaction solution. Go to the position where the DNA synthesis reaction takes place. Utilizing the pressure difference between the inlet side reaction liquid holding part 226A and the outlet side reaction liquid holding part 226B, protons are temporarily pushed through the through-hole 210a toward the place where the DNA synthesis reaction is performed. Also good. As a result, the acidic region 40 reaches the position where the DNA synthesis reaction is performed, that is, the peripheral region of the porous member 212 that closes the opening of the left through hole 210a, and protects the base 26 present in the acidic region 40. When the group 24 reacts with a proton, the protecting group 24 is removed from the base 26, and a base 26 that can react with another base is generated.

  Next, as shown in FIG. 7 (d), a second base 28 (this example) to be reacted with the reactable base 26 using a reaction liquid supply unit (not shown in FIG. 7 (d)). Then, when the amidite solution 14 containing adenine (A) is flowed, the second base 28 having a protecting group is bonded to the first base 26 from which the protecting group 24 has been released, and DNA elongation occurs. On the other hand, since the first base 26 held in the porous member 212 covering the section not irradiated with light, for example, the opening of the right side through-hole 210a in FIG. The second base 28 cannot bind and DNA elongation does not occur.

  In this way, as shown in FIG. 7E, a new base (in this example, the first base 26 present in the left porous member 212 in this example) is newly added (in this example, The second base 28) can be bound to selectively cause a DNA elongation reaction.

  Further, after the reaction system including the DNA array substrate 210 is washed with the washing solution, the cycle shown in FIGS. 7A to 7E is repeated again, so that the desired position on the DNA array substrate 210 is obtained. DNA having a desired sequence and length can be formed. In this case, if a plurality of through holes 210a are arranged in an array on the DNA array substrate 210, a DNA array in which a plurality of types of DNAs having desired sequences and lengths are arranged in an array is manufactured. Can do.

  The DNA synthesis method according to the first embodiment and the second embodiment described above will be described more specifically with reference to examples of usable reagents.

  Synthetic chemistry of DNA continues to be developed and there are various methods. A 4-step process using phosphoramide is usually used. In general, acid TCA is supplied as a method for removing the protecting group, but in this embodiment, proton is supplied by photoreaction instead of supplying acid TCA to remove the protecting group. Use a proton supply material.

  Specifically, first, 2′-deoxynucleoside protected with 5′-dimethyloxyltril is arranged on the surface of a glass-based substrate with an alkylamino linker. In addition to this, a material obtained by bonding the above 2'-deoxynucleoside protected with 5'-dimoxytritl to a polystyrene substrate is often used.

  As the proton supply material (PGA), as described above, triaryl-sulfonium-hexafluroantimonate dissolved in polypropylene carbonate can be used. In this case, the irradiation wavelength is 365 nm.

Next, after washing with CH ₃ CN, CH ₂ Cl ₂ , an extension reaction is performed in a tetrazole / CH ₃ CN solution.

After performing the extension reaction, it is washed again with CH ₃ CN and capped with a mixed solution of acetic anhydride / lutidene / THF and N-methylimidazole / THF. Next, after washing, oxidation is performed with I ₂ / THF / pyridine / H ₂ O. Thus, one cycle is completed by washing, capping, and washing.

  As mentioned above, although preferred embodiment of this invention was described referring an accompanying drawing, it cannot be overemphasized that this invention is not limited to this example. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the claims, and these are naturally within the technical scope of the present invention. Understood.

  For example, in the above-described embodiment, the case where the organic compound synthesizer is a DNA array synthesizer has been described. However, not only the synthesis of RNA but also the synthesis of proteins and general polymer compounds are also described in this embodiment. The invention can be applied.

It is sectional drawing which shows the whole structure of the DNA array synthesizer which concerns on the 1st Embodiment of this invention. It is sectional drawing which shows the structure of the DNA array substrate which concerns on the same embodiment. It is a perspective view which shows the structure of the light irradiation apparatus which concerns on the same embodiment. It is explanatory drawing which shows the synthesis | combining method of the DNA array which concerns on the embodiment, (a) is the state before the elongation reaction of DNA, (b) is the state from which the protecting group was removed, (c) is the elongation of DNA The state during the reaction, (d) shows the state after the DNA elongation reaction. It is sectional drawing which shows the whole structure of the DNA array synthesis apparatus which concerns on the 2nd Embodiment of this invention. It is sectional drawing which shows the structure of the DNA array substrate which concerns on the embodiment, (a) shows the 1st example, (b) shows the 2nd example, (c) shows the 3rd example. It is explanatory drawing which shows the synthetic | combination method of the DNA array which concerns on the embodiment, (a) is the state before the elongation reaction of DNA, (b) is the state in which the acidic region was formed by photoreaction, (c) is , A state in which the acidic region has moved and the protecting group has been removed, (c) shows a state during the DNA extension reaction, and (d) shows a state after the DNA extension reaction.

Explanation of symbols

12 Reaction Solution Containing Proton-Supplying Substance 14 Amidite Solution 22 Linker 24 Protecting Group 26 First Base (T)
28 Second base (A)
30 Photoreactive substances (proton supply substances)
40 acidic region 100 DNA array synthesizer 110 DNA array substrate 112 recess 120 reaction vessel 122 reaction solution inlet 124 reaction solution outlet 126 reaction solution holding unit 130 light irradiation device 132 light source 134 light source array substrate 136 rod lens 138 rod lens array 200 DNA array Synthesizer 210 DNA array substrate 210a Through hole 212 Porous member 220 Reaction vessel 222 Reaction liquid inlet 224 Reaction liquid outlet 226A Inlet side reaction liquid holding part 226B Outlet side reaction liquid holding part 230 Light irradiation device 232 Light source 236 Rod lens

Claims (15)

An organic compound synthesizer for synthesizing an organic compound composed of one or more repeating units by light irradiation:
A reaction vessel in which the repeating unit whose terminal is protected with a protecting group and a photoreactive substance that generates a photogenerating substance that releases the protecting group by photoreaction are supplied, and a polymerization reaction of the repeating unit is performed;
A light irradiation device having one or more light sources and irradiating light from the light sources into the reaction vessel;
An organic compound synthesizer characterized by comprising:   2. The organic compound synthesizer according to claim 1, wherein the light irradiation device synthesizes one or more organic compounds by moving relative to the reaction vessel. 3.   The light irradiation device selectively irradiates light to some of the photoreactive substances among the photoreactive substances supplied into the reaction container when moving relative to the reaction container. The organic compound synthesizer according to claim 2.   The organic compound synthesizer according to any one of claims 1 to 3, wherein the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance are performed at substantially the same position.   The organic compound synthesizer according to any one of claims 1 to 3, wherein the polymerization reaction of the repeating unit and the photoreaction of the photoreactive substance are performed at spaced positions.   6. The organic compound synthesizer according to claim 1, wherein the light irradiation device has two or more light sources arranged in an array.   The organic compound synthesizer according to claim 6, wherein the light irradiation device relatively moves in a direction perpendicular to an axial direction of the light source array.   The organic compound synthesizer according to claim 1, wherein the light source is a light emitting diode.   The organic compound synthesizer according to any one of claims 1 to 8, wherein the light irradiation device further includes one or more condensing lenses for condensing light emitted from the light source. The photoreactive substance is a proton supply substance that releases protons by photoreaction,
The organic compound synthesizer according to any one of claims 1 to 9, wherein the protecting group is a functional group capable of leaving the repeating unit by reacting with a proton.   An organic compound synthesis substrate on which an organic compound synthesized using the organic compound synthesis apparatus according to claim 1 is fixed. A plurality of photoreactive substance holding parts for holding the photoreactive substance;
The substrate for organic compound synthesis according to claim 11, wherein the polymerization reaction of the repeating unit is performed in the photoreactive substance holding unit holding the photoreactive substance irradiated with light. Having a structure capable of transmitting the repeating unit and the photogenerating material;
The photoreaction of the photoreactive substance is performed on the surface of the organic compound synthesis substrate that is irradiated with light,
The polymerization reaction of the repeating unit is performed at a position where the photoreaction of the photoreactive substance is performed on the surface of the organic compound synthesis substrate opposite to the side irradiated with light. Item 12. The organic compound synthesis substrate according to Item 11. A plurality of through holes,
In the through hole, the opening on the side irradiated with light is opened, and the opening on the side opposite to the side irradiated with light is blocked by a reactive substance transmitting member capable of transmitting the repeating unit and the photogenerating substance. And
The photoreaction of the photoreactive substance is performed in the vicinity of the opening on the side irradiated with light,
The polymerization reaction of the repeating unit is performed on the surface of the reactant transmitting member that closes the opening on the side opposite to the side irradiated with the light of the through-hole in which the photoreaction of the photoreactive material has been performed. The organic compound synthesis substrate according to claim 11, wherein the substrate is an organic compound synthesis substrate. The photoreactive substance is a proton supply substance that releases protons by photoreaction,
The substrate for organic compound synthesis according to any one of claims 11 to 14, wherein the protecting group is a functional group capable of leaving the repeating unit by reacting with a proton.

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