JP6158042B2 - Nucleic acid analysis substrate and nucleic acid analysis flow cell - Google Patents

Description

  The present invention relates to a nucleic acid analysis substrate and a nucleic acid analysis flow cell.

  Patent Document 1 discloses a substrate used in these nucleic acid analysis methods. Spots to which the sample DNA binds are arranged in a grid at a constant pitch on the substrate. The spot diameter is 300 nm. A silicon wafer is used as the substrate, and is made by a photolithography technique and an etching technique. Specifically, after forming an HMDS (Hexamethyldisilizane) layer on a silicon wafer, a positive resist is applied. After an opening is provided at a predetermined position of the resist using a photolithography technique, HMDS at the bottom of the opening is removed using oxygen plasma. Thereafter, the wafer is held in the aminosilane gas phase, and aminosilane is introduced into the bottom of the opening. After applying a resist on the wafer, dicing is performed to cut out the substrate. After removing the resist on the cut-out substrate by ultrasonic cleaning using an organic solvent, a cover glass is pasted through a polyurethane adhesive to produce a flow cell for nucleic acid analysis. By using aminosilane, which is highly hydrophilic and capable of DNA immobilization, as a DNA ball immobilization spot, prevents adsorption of DNA in other areas, and uses HMDS, which has high water repellent properties. It is possible to fix the DNA ball only on the spot.

US Patent Application Publication No. US2009 / 0270273

  The method of Patent Document 1 has a problem that the cost of the nucleic acid analysis substrate is high because an expensive silicon wafer is used as a starting material. As a method for reducing the cost, it is effective to use a synthetic resin for the substrate. However, since the synthetic resin substrate is not as flat as the silicon wafer, it is difficult to perform photolithography on the synthetic resin substrate, and it is difficult to form a minute pattern having a diameter of 300 nm. A nucleic acid analysis substrate for solving the above-described problems of the present invention is provided.

  The nucleic acid analysis substrate of the present invention comprises a synthetic resin substrate and a sample fixing spot arranged in a grid, and an inorganic oxide is provided between the sample fixing spot and the synthetic resin substrate. It is characterized by being.

  According to the present invention, a minute pattern can be formed even if a synthetic resin substrate is used.

It is a figure for demonstrating an example of a structure of the flow cell for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the board | substrate for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the board | substrate for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the board | substrate for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the flow cell structure for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the flow cell structure for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the nucleic acid analyzer of this invention.

As a result of earnest research, the inventors of the present invention use a low-cost synthetic resin substrate to form a 300 nm minute pattern on the substrate surface, introduce a functional group capable of immobilizing DNA, and contact a solution containing a DNA sample. DNA samples can be naturally fixed on minute patterns, no fluorescence emission from the synthetic resin substrate, high light transmission, no DNA or enzyme adsorption, and no substrate deflection due to temperature increase and decrease during reaction The substrate was completed.
In the present invention, a first synthetic resin substrate and a spot made of the first inorganic oxide on the first synthetic resin substrate are arranged in a grid shape, and a sample is placed on the spot made of the first inorganic oxide. A fixing spot is provided. By introducing an inorganic oxide onto the synthetic resin substrate, a functional group capable of immobilizing DNA can be introduced onto the inorganic oxide.
In the present invention, the first synthetic resin substrate is selected from the group consisting of a clereolefin polymer resin, a polycarbonate resin, a polyimide resin, an adamantate resin, and a mixture of at least two of them. . Since these resins have low DNA adsorption and high hydrophobicity, when a solution containing a DNA ball is introduced onto a substrate in combination with a functional group introduced on an inorganic oxide, the DNA ball is naturally only on the spot. Can be introduced. Further, the warpage of the substrate due to the increase in temperature during the reaction with high heat resistance can be reduced.
The present invention also provides a first synthetic resin substrate, a film made of the first inorganic oxide on the first synthetic resin substrate, and grids for fixing the sample on the film made of the first inorganic oxide. The region having no sample fixing spot is covered with a hydrophobic film. A polyvalent organic compound, a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, a salt thereof, and at least one of these compounds introduced onto a hydrophobic membrane on a run coupling agent or silane coupling agent By selecting a group consisting of two or more types of mixtures, it is possible to prevent adsorption of DNA and enzymes.
The present invention also provides a grid of spots made of silica on the first synthetic resin substrate, the film made of the second inorganic oxide on the first synthetic resin substrate, and the film made of the second inorganic oxide. A sample fixing spot is provided on a spot made of silica, and a region having no sample fixing spot on the film made of the second inorganic oxide oxide is covered with a hydrophobic film. And Etching spots made of silica by using titania, zirconia, alumina, zeolite, vanadium pentoxide, sapphire, tungsten oxide, tantalum pentoxide, and a mixture of at least two of the second inorganic oxide oxides Since the second inorganic oxide can be used as an etching stop layer when formed by etching (FIG. 4H), the end point of the etching time can be easily managed, and the first synthetic resin substrate during etching is etched. Can be prevented. In addition, a compound selected from the group consisting of a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, a salt thereof, and a mixture of at least two kinds of these compounds is used for the hydrophobic film. Since these compounds react specifically with the second inorganic oxide oxide without reacting with the fixing spot silica, a hydrophobic film is introduced only on the second inorganic oxide surface. (Figure 4I). By introducing the silane coupling agent after the introduction of the hydrophobic film or the polyvalent organic compound after the introduction of the silane coupling agent, the sample fixing spot can be introduced on the silica spot.

  That is, the present invention is as follows.

[1] a first synthetic resin substrate;
Place the spots made of the first inorganic oxide on the first synthetic resin substrate in a grid,
A nucleic acid analysis substrate comprising a spot for fixing a sample on a spot made of the first inorganic oxide.

  [2] The nucleic acid analysis according to [1], wherein the first synthetic resin substrate is selected from the group consisting of a clereolefin polymer resin, a polycarbonate resin, a polyimide resin, an adamantate resin, and a mixture of at least two of them. substrate.

  By adopting the configuration described in [2], the problem that “the synthetic resin substrate has fluorescence emission derived from the substance contained in the synthetic resin and becomes a noise of fluorescence detection during nucleic acid analysis” is solved. Can do.

[3] a first synthetic resin substrate;
A first inorganic oxide film on the first synthetic resin substrate;
Sample fixing spots are arranged in a grid on the film made of the first inorganic oxide,
A substrate for nucleic acid analysis, wherein a region having no sample fixing spot on a film made of the first inorganic acid oxide is covered with a hydrophobic film.

  [4] The first inorganic acid oxide is selected from the group consisting of silica, titania, zirconia, alumina, zeolite, vanadium pentoxide, sapphire, tungsten oxide, tantalum pentoxide, and a mixture of at least two of them. [1] The substrate for nucleic acid analysis according to [3].

  [5] A silane coupling agent or a polyvalent organic compound having a hydrophobic film introduced on the silane coupling agent, a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, their salts, and their The substrate for nucleic acid analysis according to [3] to [4], which is selected from the group consisting of a mixture of at least two kinds of compounds.

[6] a first synthetic resin substrate;
A film made of the second inorganic oxide on the first synthetic resin substrate;
The silica spots are arranged in a grid pattern on the second inorganic oxide film,
A sample fixing spot is provided on the silica spot.
A substrate for nucleic acid analysis, wherein a region having no sample fixing spot on a film made of the second inorganic acid oxide is covered with a hydrophobic film.

  [7] The second inorganic acid oxide is selected from the group consisting of titania, zirconia, alumina, zeolite, vanadium pentoxide, sapphire, tungsten oxide, tantalum pentoxide, and a mixture of at least two of them [6 ] The nucleic acid analysis substrate according to any one of the above.

  [8] The hydrophobic membrane is selected from the group consisting of a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, a salt thereof, and a mixture of at least two of these compounds [6] to [7 ] The nucleic acid analysis substrate according to any one of the above.

  [9] The nucleic acid analysis substrate according to [6] to [8], wherein the sample fixing spot is made of a silane coupling agent or a polyvalent organic compound introduced onto the silane coupling agent.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings.

  Here, in order to have a complete understanding of the present invention, specific embodiments will be described in detail, but the present invention is not limited to the contents described herein. In addition, the embodiments can be appropriately combined, and the present specification also discloses the combination form.

FIG. 1 shows an example of a reaction flow cell for nucleic acid analysis of the present invention.
In the flow cell, a first synthetic resin substrate 101, a hollow sheet 102 having a hollow portion 103 in which a central portion is cut out, and a second synthetic resin substrate 104 are bonded together. A hollow portion surrounded by the hollow portion 103, the first synthetic resin substrate 101, and the second synthetic resin substrate 104 serves as a flow path. The inlet 107 and the outlet 108 serve as a liquid inlet / outlet. In the embodiment of FIG. 1, the holes of the first synthetic resin substrate 101 are the inlet 107 and the outlet 108, but in another form, the holes of the second synthetic resin substrate are the inlet and the outlet. It may be. As yet another embodiment, holes formed in the side surface of the hollow sheet 102 may serve as an inlet and an outlet.

  A cross-sectional view of the flow cell for nucleic acid analysis of the present invention is shown (FIG. 5). The cross-sectional view is taken along the line AA ′ of FIG. FIG. 5A shows a structure in which the first synthetic resin substrate, the hollow sheet, and the second synthetic resin substrate in FIG. 1 are bonded together. In addition, the flow cell of the present invention may have a structure (FIG. 5B) in which the second synthetic resin substrate 504b in which the hollow sheet 502 and the second synthetic resin substrate 504a are integrated is bonded.

The resin used for the first synthetic resin substrate 101 is not particularly limited, and phenol resin, epoxy resin, melamine resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, transparent polyimide, polyethylene, polypropylene, poly Vinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin, polyamide, nylon, polyacetal, polycarbonate, modified polyphenylene ether, polybutylene terephthalate, polyethylene terephthalate, glass fiber reinforced polyethylene terephthalate, cyclic polyolefin, Polyphenylene sulfide, polysulfone, polyethersulfone, amorphous polyarylate, liquid crystal polymer, polyetheretherketone, and these Mixed material or glass fiber or carbon fiber reinforced inorganic material or a metal material such as a or the like can be used inside a composite material in which finely dispersed.
Generally, in the nucleic acid analysis reaction, the temperature is raised and lowered in the range of 15 ° C to 90 ° C. A resin having low heat resistance causes the substrate to be warped during this temperature change and deviates from the depth of focus of the detection system. Therefore, there is a problem that the number of spots that can be observed within one field of view is reduced and throughput is lowered. In addition, the adhesiveness between the spot 214 made of the first inorganic oxide, the film 309 made of the first inorganic oxide, and the film 418 made of the second inorganic oxide is low, and the There is also a problem that spots and films are peeled off. In addition, there are some which have a low ability to prevent the adsorption of DNA and enzymes, and there are those which contain a fluorescent substance in the resin itself and this lowers the detection sensitivity. As a material having high heat resistance, high adhesion to inorganic oxides, preventing adsorption of DNA and enzymes, and low fluorescence intensity of the resin itself, transparent polyimide, polycarbonate, or cyclic polyolefin is particularly preferable. Examples of transparent polyimide include Neoprim L (Mitsubishi Gas Chemical Co., Ltd.) and Lucera Film (JSR Corporation). Polycarbonates include SD Polyca (Sumitomo Dow), APEC HT (Bayer MaterialScience), Psychoroy (SABIC Innovative Plastics Japan), Teflon (Idemitsu Kosan), Novalex (Mitsubishi Engineering Plastics). And Lexan (SABIC Innovative Plastics Japan), Panlite (manufactured by Teijin Limited), and the like. Examples of the cyclic polyolefin include TOPAS (manufactured by TOPAS Advanced Polymers), ZEONOR (Nippon Zeon Corporation), and ARTON (manufactured by JSR Corporation).

  The thickness of the first synthetic resin substrate 101 is not particularly limited. However, in the case where the first synthetic resin substrate itself is directly installed in the temperature control unit in the nucleic acid analyzer, in order to increase the thermal conductivity. It is preferably 10 mm or less, and particularly preferably 1 mm or less.

  The hollow sheet 102 is not particularly limited as long as it can be bonded or bonded to the first synthetic resin substrate 101 and the second synthetic resin substrate 104. Examples include pressure-sensitive double-sided tapes, heat-sensitive double-sided tapes, and UV curable double-sided tapes. In a structure in which the inorganic oxide film 623 on the surface of the synthetic resin substrate and the hollow sheet 602 are in direct contact (FIG. 6), silicon rubber or the like that can be bonded to the inorganic oxide may be used. Examples of the pressure-sensitive double-sided tape include Hibon (manufactured by Hitachi Chemical Co., Ltd.), No. 500 double-sided tape (manufactured by Nitto Denko Corporation), and woven fabric double-sided adhesive tape 9075 (manufactured by Sumitomo 3M Co., Ltd.). Examples of the heat-sensitive double-sided tape include a double-sided tape for fixing abrasive cloth (manufactured by Sekisui Chemical Co., Ltd.) and a double face (manufactured by Toyochem Co., Ltd.). Examples of the UV curable double-sided tape include Dexerials (manufactured by Sony Chemical Co., Ltd.), DC1000UV (Ikkos Co., Ltd.), and the like.

  The thickness 102 of the hollow sheet is not particularly limited, but if the sheet is thin, the flow path portion surrounded by the hollow sheet portion 103, the first synthetic resin substrate 101, and the second synthetic resin substrate 104. Since the volume of the reagent used in the nucleic acid analysis reaction can be reduced, the cost of analysis can be reduced. Such a thickness is desirably 0.1 mm or less.

  The second synthetic resin substrate 102 constituting the flow channel part also serves as a detection window for detecting a nucleic acid analysis reaction performed in the flow channel. In general, since fluorescence is used for nucleic acid detection, a transparent resin that easily transmits light having an excitation wavelength or emission wavelength of a fluorescent dye is desirable. Thus, by making the second synthetic resin substrate “transparent resin”, it is possible to solve the problem that “the synthetic resin substrate has low light transmittance and low fluorescence detection sensitivity”.

  The excitation wavelength and emission wavelength of a general fluorescent dye are in the range of 450 nm to 750 nm. As such transparent resin that easily transmits the wavelength of light, transparent polyimide, polycarbonate, cyclic polyolefin, or the like is particularly preferable. Examples of transparent polyimide include Neoprim L (Mitsubishi Gas Chemical Co., Ltd.) and Lucera Film (JSR Corporation). Polycarbonates include SD Polyca (Sumitomo Dow), APEC HT (Bayer MaterialScience), Psychoroy (SABIC Innovative Plastics Japan), Teflon (Idemitsu Kosan), Novalex (Mitsubishi Engineering Plastics). And Lexan (SABIC Innovative Plastics Japan), Panlite (manufactured by Teijin Limited), and the like. Examples of the cyclic polyolefin include TOPAS (manufactured by TOPAS Advanced Polymers), ZEONOR (Nippon Zeon Corporation), and ARTON (manufactured by JSR Corporation).

  The sample fixing spot 106 is a spot for fixing the sample DNA. The sample DNA is preferably a long chain molecule in which a DNA to be measured is cut into a certain length as a starting material, and a plurality of measurement target sequence parts are copied using these cut samples as templates. As such sample DNA, non-patent literature (Science 2010, Vol. 327, pp. 78-81) discloses a DNA nanoball. When a plurality of types of DNA nanoballs are fixed to one sample fixing spot 106, a plurality of types of fluorescent dyes are detected from the one sample fixing spot 106, resulting in false detection. On the other hand, when the sample fixing spot 106 is small, the contact establishment with the DNA ball in the solution is lowered, the number of spots where the DNA ball is not fixed is increased, and the throughput of DNA base sequencing is lowered. Accordingly, the diameter of the sample fixing spot 106 is preferably such that only one DNA ball is fixed and the largest size is 50 nm or more and 500 nm or less.

  The material for the sample fixing spot 106 needs to be able to fix DNA, and to obtain a good adhesive force with the inorganic oxide existing between the sample fixing spot and the first synthetic resin substrate resin substrate. As such a compound, a silane coupling agent or a compound obtained by reacting a divalent compound with a functional group introduced by a silane coupling agent treatment is preferable. Examples of the divalent compound include those having NHS-ester group, imide ester group, sulfidyl group, epoxy group, hydrazide group and the like in the molecule. Moreover, you may introduce | transduce a functional group on an inorganic oxide using Lewis base compounds, such as a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, or those salts. Further, similarly to the silane coupling agent, the introduced functional group may be treated with a divalent compound to introduce a further functional group. The functional group introduced to the spot surface can fix the DNA through interaction with a functional group in the DNA molecule or through a covalent bond.

  The nucleic acid analysis base material of the present invention (FIG. 2H) is characterized by a structure in which a sample fixing spot 206 is provided on a spot 214 made of the first inorganic oxide on the first synthetic resin substrate 201. Since the first synthetic resin substrate 201 is in contact with the flow path, the first synthetic resin substrate 201 is required to have a high ability to prevent adsorption of DNA and enzymes. As such a material, transparent polyimide, polycarbonate, cyclic polyolefin, or the like is particularly preferable.

  In the manufacturing method, a film 209 made of a first inorganic oxide is formed on the first synthetic resin substrate 201 by using a vapor deposition method, a CVD method, a sol-gel method, or the like (FIG. 2B). As the material of the inorganic oxide, it is preferable that the first synthetic resin substrate 201 and the spot-forming film 210 have good adhesion, and such materials include titania, zirconia, alumina, zeolite, vanadium pentoxide. , Silica, sapphire, tungsten oxide, tantalum pentoxide and mixtures thereof. Next, after forming a spot forming film 210 (FIG. 2C), a pattern forming material 211 is applied (FIG. 2D). The spot forming film 210 is a film for forming the sample fixing spot 206, and as such a film, the above-described silane coupling agent or a functional group introduced by the silane coupling agent treatment is divalent. A functional group introduced on an inorganic oxide using a Lewis base compound such as a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, or a salt thereof, As in the case of the silane coupling agent, a compound obtained by treating the introduced functional group with a divalent compound and introducing a further functional group is used. The pattern forming method is preferably a nanoimprint method (FIG. 2E), which is advantageous in terms of cost. Examples of the pattern forming material 211 used in the nanoimprint method include PMMA, heat-curable UN-NIL (manufactured by Daicel Chemical Industries), and light-curing PAK-01 (manufactured by Toyo Gosei Co., Ltd.). After the pattern formation (FIG. 2F), oxygen ashing or dry etching is performed to remove the spot formation film 210 and the pattern formation material 211 in the region other than the spot formation portion (FIG. 2G). Thereafter, with the substrate immersed in an organic solvent, ultrasonic cleaning is performed to remove the pattern forming material 211 (FIG. 2H).

  In this example, patterning is performed after forming the spot forming film 210 to produce the sample fixing spot 206. However, after preparing the spot 214 made of the first inorganic oxide, the sample fixing spot 206 is prepared. Also good.

  In the base material for nucleic acid analysis of the present invention (FIG. 3H), the film 309 made of the first inorganic oxide exists on the first synthetic resin substrate 301, and the sample is formed on the film 309 made of the first inorganic oxide. The fixing spot 306 is present, and the hydrophobic film 317 is present in a region where the sample fixing spot 306 is not present. When the hydrophobic membrane 317 is introduced (FIG. 3H), if the hydrophobic membrane 317 is introduced onto the sample fixing spot 306, the ratio of the sample fixing spot 306 to which the DNA sample is fixed decreases. An optimum combination of an inorganic oxide film material that can introduce the hydrophobic film 309 only on the surface of the film 309 made of an inorganic oxide, a sample fixing spot material, and a hydrophobic film material is necessary.

  In the manufacturing method, a film 309 made of the first inorganic oxide is formed on the first synthetic resin substrate 301 (FIG. 3B). The material of the film 309 made of the first inorganic oxide is particularly preferably titania, zirconia, alumina, zeolite, or a mixture thereof. Since these inorganic materials specifically react with Lewis base compounds such as carboxylic acid compounds, phosphoric acid compounds, sulfuric acid compounds, nitrile compounds, or salts thereof, sample fixing spots 306 are selected from these materials. By selecting a material that does not react with the material to be formed, the hydrophobic film 309 can be introduced only on the surface of the film 309 made of the first inorganic oxide. Next, a sample fixing spot 306 is prepared according to the method described in non-patent literature (Langmuir 2010, Vol. 26, pp. 5603-5609). After the spot ink 316 containing aminosilane is held on a PDMS (Polydimethylsiloxane) stamper 315 (FIG. 3C), this is transferred to a film made of a first inorganic oxide to produce a sample fixing spot 306 (FIG. 3). 3D to Figure 3G). Thereafter, the substrate is immersed in an aqueous solution containing a Lewis base compound such as a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, or a salt thereof, and a hydrophobic film is formed on the film 309 made of the first inorganic oxide. Introduce 317. As such a compound, a compound having a hydrophobic site such as a long-chain alkyl in the molecule is desirable. Examples of carboxylic acid compounds that can be used to form the blocking layer include sodium iminodiacetate, ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid, ethylene glycol-bis (2-aminoethyl ether) -N, N, N ′, N′-tetraacetic acid, 10 -Carboxy-1-decanethiol, 7-carboxy-1-heptanethiol, 5-carboxy-1-pentanethiol, 10-carboxyl disulfide, 7-carboxyl heptyl disulfide, 5-carboxyl pentyl sulfide, 15-carboxyl-1-pentadecane Examples include thiol, carboxy-EG6-undecanethiol, carboxy-EG6-hexadecanethiol, butyl formate, citronellyl formate, ethyl formate, geranyl formate undecanoic acid, and salts thereof. Examples of phosphoric acid compounds include glyceraldehyde 3-sphingosine phosphate, 1-deoxy-D-xylulose-5-phosphate, 3-sn-phosphatidic acid, O-phosphorylethanolamine dihydrogen phosphate Examples include 2-aminoethyl and salts thereof. Examples of the sulfuric acid compound include sodium decyl sulfate, sodium dodecyl sulfate, and sodium octyl sulfate. Examples of the nitrile compound include dicyanopentane, dicyanohexane, (butylamino) acetonitrile, (dimethylamino) propionitrile, and hexynenitrile.

  In the base material for nucleic acid analysis of the present invention (FIG. 4J), a film 418 made of the second inorganic oxide is present on the first synthetic resin substrate 401, and silica is formed on the film 418 made of the second inorganic oxide. A spot 421 made of silica, a spot 406 for fixing the sample on the spot 421 made of silica, and a hydrophobic film 420 in a region where the spot 406 for fixing the sample does not exist. During etching of the first inorganic oxide (FIG. 4G), since the film 418 made of the second inorganic oxide serves as an etching stop layer, it is possible to prevent the first synthetic resin substrate from aggravating the resin substrate due to overetching. This can increase the manufacturing yield during mass production.

  A film 418 made of the second inorganic oxide is formed on the first synthetic resin substrate 401 (FIG. 4B). The material of the film 418 made of the second inorganic oxide is particularly preferably titania, zirconia, alumina, zeolite, or a mixture thereof. Since these inorganic materials specifically react with Lewis base compounds such as carboxylic acid compounds, phosphoric acid compounds, sulfuric acid compounds, nitrile compounds, or salts thereof, spot 421 made of silica is selected from these materials. By selecting a material having low reactivity with the material to be formed, the hydrophobic film 420 can be introduced only on the surface of the film 418 made of the second inorganic oxide without reacting with the spot 421 made of silica. (Figure 4I).

  Thereafter, a film 419 made of silica is introduced. Since silica has low reactivity with Lewis base compounds such as carboxylic acid compounds, phosphoric acid compounds, sulfuric acid compounds, nitrile compounds, or salts thereof, the Lewis base on the spot 421 made of silica when the hydrophobic film 420 is introduced The introduction of the compound can be prevented.

Next, after applying the pattern forming material 411, a spot forming portion is manufactured using a stamper (FIGS. 4D to 4F). Thereafter, using a dry etching process, the film made of the pattern forming material 411 and silica in the region other than the spot forming portion is removed (FIG. 4G). At this time, by using a mixed gas of CF4 and CHF or a mixed gas of SF6 as a reactive gas in the dry etching process, the film 418 made of the second inorganic oxide can be used as an etching stop layer.
Next, after removing the pattern forming material using oxygen ashing or the like (FIG. 4H), the substrate is made of a Lewis base compound such as a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, or a salt thereof. The water-repellent membrane 420 is introduced by immersing in (FIG. 4I). Finally, the substrate is treated with a silane coupling agent, and a sample fixing spot 406 is introduced onto the silica spot 421.

  The thus prepared flow cell for nucleic acid analysis is set in a nucleic acid analyzer and subjected to nucleic acid analysis. The nucleic acid analyzer 724 includes a nucleic acid analysis flow cell 725 of the present invention; a nucleic acid analysis flow cell 725 fixed; a holder unit 726 capable of adjusting the temperature of the nucleic acid analysis flow cell 725; and a nucleic acid analysis flow cell 725. And a stage unit 727 for moving the holder unit 726; a reagent container 728 containing a plurality of reagents and washing water; and a drive source for aspirating and injecting the reagent contained in the reagent container 728 into the flow cell 725 for nucleic acid analysis A liquid feeding unit 729; a nozzle 730 that actually accesses the reagent container 728 and the nucleic acid analysis flow cell 725 when aspirating and discharging the reagent; a nozzle conveyance unit 731 that conveys the nozzle 730; a nucleic acid analysis flow cell 725 A detection unit 732 for observing the sample fixed to the liquid, and a waste liquid container 733 for storing the waste liquid. The detection unit includes, for example, a fluorescence detection device, and can irradiate a flow cell for nucleic acid analysis on the stage unit with excitation light to detect generated fluorescence.

  The nucleic acid analyzer operates as follows. First, a flow cell 725 for nucleic acid analysis holding a nucleic acid sample to be measured is installed in the holder unit 726. Next, the nozzle 730 accesses the reagent container 728 and sucks the reagent by the liquid feeding unit 729. The nozzle 730 is transported to the upper surface of the nucleic acid analysis flow cell 725 by the nozzle transport unit 731 and injects the reagent into the nucleic acid analysis flow cell 725. The holder unit 726 reacts by adjusting the temperature of the nucleic acid sample and the reagent contained in the flow path of the nucleic acid analysis flow cell 725. In order to observe the reacted nucleic acid sample, the nucleic acid analysis flow cell 725 is moved by the stage unit 727, and excitation light is applied to detect fluorescence of a plurality of nucleic acid samples in the detection region. In this case, it is preferable to detect fluorescence by applying excitation light through a nucleic acid sample or a substrate on which a carrier having a nucleic acid sample on the surface is fixed. After the detection, the operation of moving the nucleic acid analysis flow cell 725 minutely and detecting in the same manner is repeated a plurality of times. When observation is completed in all detection regions, the washing water contained in the reagent container 728 is sucked by the liquid feeding unit 729 and injected into the flow cell 725 for nucleic acid analysis, thereby washing the flow path of the flow cell 725 for nucleic acid analysis. To do. Moreover, a nucleic acid sample can be read by repeating the operation | movement of inject | pouring another reagent and detecting several times. The nucleic acid analyzer is controlled by a computer and can automatically perform the above operation.

  Various nucleic acid analyzes can be performed using the nucleic acid analyzer of the present invention, and for example, DNA sequencing and hybridization can be performed. In particular, DNA sequencing (DNA sequencing) can be performed using the nucleic acid analyzer of the present invention. The method of DNA sequencing is not limited, but DNA sequencing by a method using stepwise ligation (Sequencing by Oligonucleotide Ligation and Detection) is preferable. The stepwise ligation method is a technique in which a fluorescently labeled probe is sequentially bound using a single-stranded DNA on a fine particle as a template to determine a sequence by two bases. By this method, tens to hundreds of bps can be decoded in one cycle, and tens of Gb data can be analyzed in one run. In the stepwise ligation method, parallel sequencing can be performed by repeatedly hybridizing fluorescently labeled oligonucleotides. The sequencing method using stepwise ligation can be performed, for example, as described in Science 2010, Vol. 327, pp. 78-81 (Non-patent Document 4).

A nucleic acid analysis method using the nucleic acid analyzer of the present invention will be described. The analysis method conforms to the method disclosed in Science 2010, Vol. 327, pp. 78-81.
(1) Preparation of circular library After DNA to be measured was fragmented with ultrasonic waves, a 500-base long DNA fragment was recovered by acrylamide gel electrophoresis and purified with a QiaQuick column purification kit (QIAGEN). The end of the DNA fragment was dephosphorylated using FastAP (Thermo scientific), and then the Ad1 adapter was ligated. After removing the unreacted adapter using AMPure (Beckman Coulter), PCR amplification was performed using PfuTurbo Cx (Agilent Technology). The terminal portion of the obtained amplification product was cleaved with USER (New England BioLabs), and then a part of the base of the enzyme recognition site was methylated using Eco57I (Thermo scientific). After circular DNA was synthesized using T4 DNA ligase (Enzymatics), unreacted linear DNA was cleaved and removed using PlasmidSafe exonuclease (Epicentre). The circular DNA was cleaved using AcuI (New England BioLabs) and then amplified using Taq DNA polymerase (New England BioLabs). A similar reaction was performed to synthesize linear DNA into which Ad2 adapter, Ad3 adapter, and Ad4 adapter were introduced. After amplification reaction using Pfu Turbo Cx (Agilent Technology), circular DNA was synthesized using CircLigase (Epicentre).
Finally, unreacted linear DNA was decomposed and removed using ExoI (Epicentre) and ExoIII (Epicentre).
(2) DNA ball production. DNA ball fixation to a flow cell for nucleic acid analysis A DNA library was synthesized by performing an amplification reaction using Phi29 DNA polymerase (New England BioLabs) using a circular library as a template. Thereafter, an aqueous solution containing DNA balls was poured into the flow cell for nucleic acid analysis of the present invention and held at 23 ° C. for 2 hours, and the DNA balls were fixed to the sample fixing spot 206. Finally, the inside of the flow cell was washed with neutral water to remove unfixed DNA balls.
(3) Nucleic Acid Analysis A nucleic acid analysis flow cell having a DNA ball fixed thereto was installed in the nucleic acid analyzer 725 of the present invention. An aqueous solution containing an anchor primer was injected into the flow cell 725 for nucleic acid analysis via a nozzle 730. Using the holder unit 726, the aqueous solution in the flow cell 725 for nucleic acid analysis was held at 28 ° C. for 30 minutes. An aqueous solution containing a primer and a ligation enzyme labeled with a dye for sequencing was injected, and then kept at 28 ° C. for 60 minutes. The flow path in the flow cell 725 for nucleic acid analysis was washed with a buffer to remove unreacted labeled primer. After the nucleic acid analysis flow cell 725 was moved by the stage unit 727, excitation light was applied by the detection unit 732 to detect fluorescence introduced into a plurality of DNA balls in the detection region. The DNA sequence was determined by repeating the ligation reaction, washing, and fluorescence detection for a plurality of cycles.

  Various nucleic acid reactions can be performed using the flow cell for nucleic acid analysis or the nucleic acid analyzer of the present invention, and nucleic acids such as DNA sequences can be analyzed.

101,201,301,401,501,601 First synthetic resin substrate
102,502,602 hollow sheet
103,603 hollow section
104,504a, 504b, 604 Second synthetic resin substrate
105,505a, 505b Spot grid array
106,206,306,406,606 Sample fixing spot
107,607 inlet
108,608 outlet
209,309 First inorganic oxide film
210 Film for spot formation
211,411 Pattern forming material
212,315,412 stamper
213,413 Spot formation section
214 Spot made of the first inorganic oxide
316 spot ink
317,420,622 Hydrophobic membrane
418 Film made of the second inorganic oxide
419 Silica film
421 Silica spot
623 Film comprising first inorganic oxide and second inorganic oxide
724 Nucleic acid analyzer
725 Flow cell for nucleic acid analysis
726 Holder unit
727 stage unit
728 Reagent container
729 Liquid feed unit
730 nozzle
731 Nozzle transfer unit
732 detection unit
733 Waste liquid container

Claims (5)

A method for producing a flow cell for nucleic acid analysis for producing a flow cell for nucleic acid analysis,
An inorganic oxide film made of an inorganic oxide is formed on a synthetic resin substrate,
Introducing a silica film made of silica on the inorganic oxide film,
Apply the pattern forming material, and then use the pattern forming material to produce the spot forming part,
Using an etching process, the silica film and pattern forming material other than the spot forming part are removed,
Removing the remaining patterning material, then introducing a hydrophobic film,
A method for producing a flow cell for nucleic acid analysis, wherein a sample fixing spot for fixing a sample is formed on the silica membrane. In claim 1,
For the nucleic acid analysis, the synthetic resin substrate is any one selected from the group consisting of a clereolefin polymer resin, a polycarbonate resin, a polyimide resin, an adamantate resin, and a mixture of at least two of them. A manufacturing method of a flow cell. In claim 1,
The inorganic oxide is any one selected from the group consisting of titania, zirconia, alumina, zeolite, vanadium pentoxide, sapphire, tungsten oxide, tantalum pentoxide, and a mixture of at least two of them. A method for producing a flow cell for nucleic acid analysis, which is characterized. In claim 1,
The hydrophobic membrane is any one selected from the group consisting of a carboxylic acid compound, a phosphoric acid compound, a sulfuric acid compound, a nitrile compound, a salt thereof, and a mixture of at least two of these compounds. A method for producing a flow cell for nucleic acid analysis. In claim 1,
The method for producing a flow cell for nucleic acid analysis, wherein the sample fixing spot is either a silane coupling agent or a polyvalent organic compound introduced onto the silane coupling agent.