JP2010243223A - Nucleic acid analyzing device and nucleic acid analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect the fluorescence, which is emitted from fluorescent dye by irradiation with exciting light with a wavelength of about 500 nm, with high precision in a nucleic acid analyzing device. <P>SOLUTION: In the nucleic acid analyzing device having a metal body producing localized surface plasmon by light irradiation, the material of the metal body consists of metal selected from any of ruthenium, iridium, palladium and rhodium or an alloy of the metals. These metals efficiently produce localized surface plasmon with respect to light with a wavelength of about 500 nm. Further, as the metals have extremely low ionization tendency and are very chemically stable, they can be used in a reaction solution for a long time. The present invention enables fluorescent measurement of nucleic acid stably and with high efficiency using light with the wavelength of about 500 nm. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a nucleic acid analysis device and a nucleic acid analysis apparatus.

  As a nucleic acid analysis device, a new technique for determining the base sequence of DNA or RNA has been developed.

  In the currently used method using electrophoresis, a cDNA fragment sample synthesized by reverse transcription reaction from a DNA fragment or RNA sample for sequencing is prepared in advance, and the well-known Sanger method (dideoxy method) is used. After performing the base extension reaction, electrophoresis is performed, and the molecular weight separation development pattern is measured and analyzed.

  On the other hand, in recent years, as described in “PNAS 2003, Vol. 100, pp. 3960-3964” (Non-patent Document 1), a method for fixing a DNA or the like on a substrate and determining its base sequence has been proposed. ing. Each sample DNA fragment to be analyzed is captured at a random position on the surface of the substrate, extended by about one base, and the result is detected by fluorescence measurement to determine the base sequence. Specifically, first, four types of dNTP derivatives (MdNTPs) that are incorporated into a template DNA as a substrate for DNA polymerase and can stop the DNA chain elongation reaction due to the presence of a protective group and have a label that can be detected. ) To perform a DNA polymerase reaction. Next, a step of detecting the incorporated MdNTP by fluorescence or the like and a step of returning the MdNTP to a state where it can be extended are performed. These three steps are regarded as one cycle, and the cycle is repeated to determine the base sequence of the sample DNA. In the present technology, a plurality of DNA fragments can be analyzed at the same time by increasing the reaction and fluorescence emission in parallel at a plurality of positions on the substrate, and the analysis throughput can be increased. In addition, in this method, since the base sequence can be determined with a single DNA molecule, there is no need for amplification or purification of sample DNA by cloning or PCR, which has been a problem in the prior art, and rapid genome analysis and genetic diagnosis. Can be expected.

  When analyzing a base sequence using an extension reaction on a substrate, a single base extension reaction / cleaning / measurement of an unreacted substrate as represented by the method disclosed in Non-Patent Document 1 is one cycle. A sequential reaction system is generally used. When analyzing the base sequence for each single DNA molecule, the fluorescence of one molecule of the fluorescent dye attached to the nucleotide incorporated into the DNA duplex by the single base extension reaction on the probe DNA is measured. However, in a normal fluorescence measurement, it is not possible to discriminate between a fluorescent molecule captured on the probe DNA and a fluorescent dye associated with an unreacted nucleotide floating in the vicinity thereof. Therefore, it was indispensable to wash the unreacted substrate when the base was extended by one base. By entering this cleaning process, it is necessary to form complicated flow paths, liquid feeding devices, and waste liquid treatment devices on the substrate, a large amount of reaction reagent is consumed, and further, it is necessary for total analysis. There was a problem that reaction time also became long.

  In order to distinguish between a single fluorescent dye molecule captured on the probe DNA and a fluorescent molecule of the unreacted substrate without washing the unreacted substrate, the fluorescence intensity from the fluorescent dye captured on the probe DNA is high. We must create conditions where the intensity from the floating dye is low.

  The use of fluorescence enhancement as disclosed in “Physical Review Letters 2006, vol. 96, pp. 113002-113005. (Non-patent Document 2)” can be considered for such a problem. When the metal body is irradiated with light, an optical near field enhanced by the localized surface plasmon near the surface of the metal body is generated. At this time, a phenomenon called fluorescence enhancement is known in which the emission intensity of the fluorescent molecules existing in the optical near field is enhanced.

P.N.A.S. 2003, Vol. 100, pp. 3960-3964. Physical Review Letters 2006, vol. 96, pp. 113002-113005 Anal. Chem. 2006, vol. 78, pp. 6238-6245 Anal. Chem. 2007, vol. 79, pp. 6480-6487 Nano Letters. 2004, vol.4, 957-961 P.N.A.S. 2006, vol. 103, pp 19635-19640 P.N.A.S. 2008, vol. 105, pp 1176-1181 P.N.A.S. 2008, vol. 105, pp 91459150

  As a result of intensive studies on the detection of weak fluorescence from a fluorescent label of a single DNA molecule, the present inventor has obtained the following knowledge.

In the analysis of biomolecules using fluorescent labels, particularly in the nucleic acid analysis as described above, it is desirable to use light in the wavelength range of 300 to 700 nm, particularly in the vicinity of the wavelength of 500 nm, as excitation light for the following reasons. .
(1) The excitation wavelength of a general fluorescent dye is about 300 to 700 nm. Although there are dyes having an excitation wavelength outside this range, fluorescent dyes having an excitation wavelength of about 300 to 700 nm may be used for reasons such as quantum yield, ease of labeling, ease of production and availability, and stability. desirable.
(2) The spectral sensitivity characteristic of a CCD mainly used as an element for detecting the emission of fluorescent molecules generally has a peak at 500 to 600 nm. The excitation wavelength of a general fluorescent dye having a fluorescence wavelength peak in this range is about 400 to 560 nm.
(3) In order to distinguish and use a plurality of fluorescent dyes, the selection of fluorescent dyes is narrowed due to the problem of the selectivity of the emission wavelength, and it is essential to use excitation light having a wavelength of around 500 nm. In nucleic acid analysis, it is necessary to distinguish four types of bases (A, T, G, C), which is particularly important.

  However, Non-Patent Document 2 discloses gold as a metal that causes fluorescence enhancement by localized surface plasmons, but the optimum excitation wavelength of gold localized surface plasmons is around 700 nm, and the wavelength is around 500 nm. Then, the fluorescence enhancement effect is very small.

  An object of the present invention relates to detection of fluorescence generated from a fluorescent dye with high accuracy by irradiation with excitation light having a wavelength of around 500 nm in a nucleic acid analysis device.

The present invention relates to a nucleic acid analysis device having a metal body that generates localized surface plasmons by light irradiation, wherein the metal body material is a metal selected from ruthenium, iridium, palladium, or rhodium, or these Regarding being an alloy. These metals efficiently generate localized surface plasmons for light having a wavelength of around 500 nm. In addition, these metals have a very small ionization tendency and are chemically very stable, so that they can be used in a reaction solution for a long time.

According to the present invention, fluorescence of a nucleic acid can be measured with high efficiency and stability using light having a wavelength of around 500 nm. For example, by increasing the fluorescence intensity from the fluorescent dye captured by the probe DNA or the like and increasing the contrast with the background light intensity from the floating fluorescent dye, It is possible to provide a nucleic acid analysis device that discriminates a fluorescent molecule of a reaction substrate and measures a base extension reaction.

1 is a schematic diagram of a nucleic acid analysis device in this example. 1 is a schematic diagram of a nucleic acid analysis device in this example. The flowchart for demonstrating an example of the manufacturing method of a nucleic acid analysis device. The flowchart for demonstrating an example of the manufacturing method of a nucleic acid analysis device. The schematic diagram in order to explain an example of a form of use of a nucleic acid analysis device. Calculation results of the effect of the nucleic acid analysis device. Schematic for demonstrating an example of the nucleic acid analyzer using a nucleic acid analysis device.

  In this example, a nucleic acid analysis device for analyzing a nucleic acid in a sample by luminescence measurement, which includes a support substrate and has a nanostructure in contact with the support substrate, is disclosed. Also disclosed are nanostructures comprising one or more metal bodies that generate localized surface plasmons in the vicinity of the metal bodies upon light irradiation, and probes for analyzing nucleic acids in the sample. It is also disclosed that the probe is disposed at a surface plasmon generation site and the metal body material is a metal selected from ruthenium, iridium, palladium, and rhodium, or an alloy thereof.

  In this example, a nucleic acid analysis device for analyzing a nucleic acid in a sample by fluorescence measurement is disclosed. By combining the optical filter of the detection device, fluorescence of two or more colors can be detected simultaneously.

  In this example, a nucleic acid analysis device in which the probe is one or more macromolecules selected from nucleic acids or proteins is disclosed. A protein such as a nucleic acid or a nucleic acid synthase has high specificity when taking up a nucleic acid to be measured, and can specifically measure only the measurement target.

  In this example, a nucleic acid analysis device in which reaction sites including nanostructures and probes are arranged in an array on a support substrate is disclosed. Since only an arbitrary reaction site on the support substrate needs to be analyzed, the configuration of the analyzer can be simplified.

  In this example, a nucleic acid analysis device is disclosed in which the probe contained in one reaction site is a single molecule. Since an amplification step for the nucleic acid to be measured is unnecessary, the analysis time can be shortened.

  In this embodiment, the nucleic acid analyzing device is provided with means for supplying one or more types of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthase, primers, and nucleic acid samples; Incorporated into the nucleic acid chain by a nucleic acid elongation reaction caused by the coexistence of one or more types of biomolecules comprising nucleotide, a nucleotide having a fluorescent dye, a nucleic acid synthase, a primer, and a nucleic acid sample on the nucleic acid analysis device A nucleic acid analyzer that includes the fluorescence detection means for measuring the fluorescence of the fluorescent dye and obtains the base sequence information of the nucleic acid sample. Due to the enhancement effect by the localized surface plasmon, an expensive optical member necessary for high-sensitivity detection becomes unnecessary.

  Further, in this example, the nanostructure includes a plurality of metal bodies facing each other on the support substrate, and localized surface plasmons are generated in the space where the metal bodies face each other by light irradiation. Disclosed is a nucleic acid analysis device in which a second metal different from a metal body is present between a substrate and a probe for analyzing a nucleic acid in a sample on the second metal. Using the difference in reactivity between the metal constituting the metal body and the second metal, the probe for measurement can be specifically arranged on the second metal.

  Further, in this example, the nanostructure includes a plurality of metal bodies facing each other on the support substrate, and localized surface plasmons are generated in the space where the metal bodies face each other by light irradiation. Disclosed is a nucleic acid analysis device having an insulator between a substrate and a probe for analyzing a nucleic acid in a sample on the insulator. A probe for measurement can be specifically arranged on the insulator by using the difference in reactivity between the metal constituting the metal body and the insulator.

  Moreover, in a present Example, it discloses that the distance between the metal bodies which exist facing each other is 15 nm or less. By reducing the distance between the metal bodies, the enhancement effect by the localized plasmon can be enhanced. Moreover, the probe fixing region for measurement can be reduced, and the proportion of the metal body in which only a single molecule is immobilized can be increased.

  Further, in this example, a support base having a metal body that generates localized surface plasmons by light irradiation is provided, and nucleic acids in a sample are arranged in a space where localized surface plasmons are generated, and fluorescence measurement is performed on the nucleic acids. In the nucleic acid analysis device analyzed by the above, it is disclosed that the metal body is composed of ruthenium.

  Further, in this example, a support base having a metal body that generates localized surface plasmons by light irradiation is provided, and nucleic acids in a sample are arranged in a space where localized surface plasmons are generated, and fluorescence measurement is performed on the nucleic acids. In the nucleic acid analysis device analyzed by the above, it is disclosed that the metal body is composed of iridium.

  Further, in this example, a support base having a metal body that generates localized surface plasmons by light irradiation is provided, and nucleic acids in a sample are arranged in a space where localized surface plasmons are generated, and fluorescence measurement is performed on the nucleic acids. In the nucleic acid analysis device analyzed by the above, it is disclosed that the metal body is composed of palladium.

  Further, in this example, a support base having a metal body that generates localized surface plasmons by light irradiation is provided, and nucleic acids in a sample are arranged in a space where localized surface plasmons are generated, and fluorescence measurement is performed on the nucleic acids. It is disclosed that the metal body is composed of rhodium in the nucleic acid analysis device that analyzes by the above.

  Further, in this example, a support base having a metal body that generates localized surface plasmons by light irradiation is provided, and nucleic acids in a sample are arranged in a space where localized surface plasmons are generated, and fluorescence measurement is performed on the nucleic acids. In the nucleic acid analysis device analyzed by the above, it is disclosed that the metal body is composed of an alloy composed of two or more of ruthenium, iridium, palladium, rhodium, and platinum.

  In this example, a nucleic acid analysis device is disclosed in which the wavelength of light irradiation for generating localized surface plasmons is 300 to 700 nm.

  Further, in this embodiment, a plurality of metal bodies are present facing each other on the support base, and localized surface plasmons are generated by light irradiation in the space where the metal bodies face each other. Disclosed is a nucleic acid analysis device in which a second metal different from a metal body is present, and a probe for analyzing a nucleic acid in a sample is disposed on the second metal.

  Further, in this embodiment, a plurality of metal bodies exist facing each other on the support base, and localized surface plasmons are generated by light irradiation in the space where the metal bodies face each other, and insulation is provided between the space and the support base. A nucleic acid analysis device is disclosed in which a body is present and a probe for analyzing a nucleic acid in a sample is disposed on the insulator.

  In this example, a nucleic acid analysis device in which the probe is one or more macromolecules selected from nucleic acids or proteins is disclosed.

  In this example, a nucleic acid analysis device in which the probe is a single molecule is disclosed.

  In this example, a nucleic acid analysis device is disclosed in which a plurality of metal bodies facing each other are arranged in an array on a support substrate.

  In this example, a nucleic acid analysis device in which the interval between the plurality of metal bodies is 15 nm or less is disclosed.

  Further, in this embodiment, a means for supplying one or more types of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthase, primers, and nucleic acid samples to the nucleic acid analysis device, and light to the nucleic acid analysis device In the nucleic acid chain by a nucleic acid elongation reaction caused by the coexistence of one or more types of biomolecules consisting of a nucleotide, a nucleotide having a fluorescent dye, a nucleic acid synthase, a primer, and a nucleic acid sample on a nucleic acid analysis device Disclosed is a nucleic acid analyzer that includes fluorescence detection means for measuring the fluorescence of an incorporated fluorescent dye and that acquires base sequence information of a nucleic acid sample.

  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. Moreover, each Example can be combined suitably and this specification is also disclosing about the combination form.

  The nucleic acid analysis device in this example will be described with reference to FIG.

  In order to distinguish between the fluorescent dye captured by the probe and the fluorescent molecule of the unreacted substrate, the intensity of light applied to the fluorescent dye captured by the probe and the floating unreacted substrate fluorescent dye is roughly divided. In other words, it is necessary to make the radiation process efficiently occur only by the fluorescent dye captured by the probe. This example is based on the latter concept, and as reported in “Physical Review Letters 2006, 96, pp 113002-113005 (Non-patent Document 2)”, localized surface plasmons are expressed as molecules. This is based on a physical phenomenon that increases the probability of both electronic transition due to light absorption and radiation transition from the excited singlet to the ground state. The fluorescence enhancement effect of localized surface plasmons can be expected to be several to several tens of times.

  In “Nano Letters. 2004, vol. 4, 957-961” (Non-Patent Document 5), when a triangular prism-shaped metal body approaches, strong localized surface plasmons may be generated in the space between them. It is shown. The closer the distance between the triangular prisms, the stronger the localized surface plasmon is generated. However, in the structure of the metal body disclosed in Non-Patent Document 5, it is difficult to specifically arrange a probe for measurement in a space where localized surface plasmons are generated. In particular, it is almost impossible to fix only one molecule at a predetermined position.

  In the nucleic acid analysis device of this example, a metal body 105 similar to a triangular prism faces on a support substrate 104, and a space 106 therebetween is a fluorescence enhancement field by localized surface plasmons. A second metal 107 different from the metal constituting the triangular prism is disposed between the space 106 where the localized surface plasmon is generated and the support base 104, and the probe 101 is fixed to the second metal 107. . As a result, strong localized surface plasmons are generated, and the probe 101 can be immobilized in the space 106 near the localized surface plasmon generation site. Then, only the fluorescent dye 102 incorporated into the probe 101 benefits from fluorescence enhancement, resulting in a difference in fluorescence intensity of several to several tens of times that of the floating unreacted fluorescent dye 103. Moreover, it is a structure which can be manufactured by utilizing a thin film process used for manufacturing semiconductors and wiring boards, and can be manufactured at low cost.

  Since the generation of a strong depolarization field (an electric field having a phase opposite to the applied electric field by light) in the metal due to the electric field caused by light leads to strong localized surface plasmon formation, the material of the metal body 105 is more absolute. A metal having a large negative complex dielectric constant real part is preferable, and specifically, ruthenium, rhodium, iridium, and palladium are preferable. These metals efficiently generate localized surface plasmons for light having a wavelength of around 500 nm. Moreover, since these metals have a very small ionization tendency and are chemically very stable, they can be used for a long time in the reaction solution. Alternatively, the characteristics can be improved by using an alloy containing ruthenium, rhodium, iridium, or palladium as the metal body 105. For example, by using an iridium-rhodium alloy containing 50% rhodium, the oxidation resistance of the metal body can be increased. Platinum rhodium can also increase the stability to oxidation. Alternatively, a plurality of types of metal thin films containing ruthenium, rhodium, iridium, and palladium can be stacked as the metal body 105.

  The second metal 107 is not particularly limited as long as the probe 101 can be specifically immobilized using a difference in chemical properties from the surface of the metal body 105. Further, a suitable functional group is selected and applied to the second metal, or the functional group in the probe 101 is reacted with the functional group further modified using the functional group or the functional group as a reactive base point. Thus, the desired probe 101 may be fixed. As such a combination of the metal body 105 and the second metal 107, if the metal body 105 is ruthenium, rhodium, iridium, palladium, or an alloy thereof, the second metal 107 is titanium, nickel, Examples thereof include at least one metal selected from chromium, iron, cobalt, cadmium, aluminum, gallium, indium, zirconia, niobium, hafnium, and tantalum, and alloys thereof. Alternatively, a conductive oxide film such as ITO may be used. A desired functional group for immobilizing the probe 101 can be introduced by reacting a carboxylic acid, a phosphonic acid, a phosphate ester, or an organosilane compound on the oxide film formed on the surface of the second metal 107. it can.

  An insulator may be provided between the space 106 and the support base 104. The insulator to be used is not particularly limited, but a material capable of forming a thin film by vapor deposition, sputtering, CVD (Chemical Vapor Deposition), PVD (Physical Vapor Deposition), or the like is desirable from the viewpoint of workability in a minute region. Such materials include silicon, titanium, beryllium, zirconium, tungsten, boron, hafnium, vanadium, tantalum, aluminum, thorium, molybdenum, iron and other carbides, nitrides, borides, silicides, or oxides. Can be mentioned.

  The functional group introduced onto the second metal 107 or the insulator is not particularly limited, but as a reactive base point for fixing the probe 101, an amino group, a thiol group, a carboxyl group, a hydroxyl group, an aldehyde group, a ketone Groups and the like. Furthermore, as a technique for improving the reaction efficiency for immobilizing the probe 101, a functional group such as NHS-ester group, imide ester group, sulfidyl group, epoxy group, hydrazide group, etc. is introduced using a divalent compound. Also good. Further, in order to increase the single molecule immobilization rate in the enhancement field, the probe 101 may be immobilized via a bulky compound such as avidin, dendron, crown ether or the like.

  The probe 101 is not particularly limited as long as it can supplement the nucleic acid 108 to be measured. Examples of probes that can directly capture the nucleic acid 108 include nucleic acids such as DNA, RNA, and PNA, and proteins such as enzymes. Alternatively, the nucleic acid 108 may be supplemented via a chromosome, nucleoid, cell membrane, cell wall, virus, antigen, antibody, lectin, hapten, receptor, peptide, sphingosaccharide, glycosphingolipid, aptamer, and the like.

  Since the resonance frequency suitable for the generation of surface plasmon is due to the interaction between the free electron group on the surface of the metal body and light, the appropriate size of the metal body 105 varies depending on the wavelength of the light to be irradiated. If the wavelength of the excitation light is 300 to 850 nm, the size of the metal body is suitably about 10 to 1000 nm in both width and height, but is not limited to this condition. The cylinders as shown in FIG. 2A are connected by prisms, and there is a space 206 in which localized surface plasmons are generated between the prisms. As shown in FIG. 2B, the cylinders are arranged in the smallest cylinder. Examples include a space 206. Further, as shown in (C), a region other than the metal body 205, the space 206, and the second metal 207 may be covered with a material 209 having a refractive index lower than that of the material constituting the support base 204. Since the unreacted fluorescent dye does not enter the region covered with the material 209 having a low refractive index, the background derived from the fluorescent dye can be reduced.

  A method for producing a nucleic acid analysis device having a probe immobilized on a second metal will be described with reference to FIG.

(1) Formation of second metal film A thin film of the second metal 307 is formed on a smooth support substrate 304. For the smooth support base 304, a glass substrate, a quartz substrate, a sapphire substrate, a resin substrate, or the like is used. When it is necessary to irradiate the excitation light from the back surface opposite to the surface on which the metal body 305 is formed, a quartz substrate or a sapphire substrate having excellent light transmittance may be used. When the second metal 307 is irradiated with excitation light from the back surface, the thickness is preferably as small as possible, and more preferably 5 to 100 nm. The thin film is made using vapor deposition, sputtering, CVD, PVD, or the like.

(2) Formation of silicon film, (3) Patterning of silicon A silicon film having a thickness of 5 nm or more is formed on the second metal 307. The thin film forming method is preferably vapor deposition, sputtering, CVD, PVD, or the like. The obtained silicon film is subjected to photolithography and etching to perform patterning for creating a space 306 in which localized surface plasmons between the metal bodies 305 are generated. The pattern conforms to a desired pattern for arranging the facing metal bodies 305 in an array. For example, when the metal bodies 305 facing each other with a pitch of 1 μm are configured, if the formation region is 1 mm × 1 mm, 1 million reaction sites can be formed. For photolithography, a method using an existing i-line (wavelength 365 nm), KrF excimer laser (wavelength 248 nm), ArF excimer laser (wavelength 193 nm), X-ray, or electron beam as a light source can be used. In order to increase the patterning accuracy of etching, it is preferable to use RIE (Reactive Ion Etching).

(4) Formation of insulating film, (5) Etching of insulating film, (6) Etching of silicon An insulator 311 is formed on silicon by using CVD. The thickness of the insulating film controls the distance between the metal bodies 305. The shorter the distance between the metal bodies 305, the higher the fluorescence enhancement effect by localized surface plasmons. A preferable thickness of the insulator 311 is 50 nm or less, more preferably 15 nm or less. The method of controlling the distance between the metal bodies 305 by the film thickness of the insulating film as in this embodiment is preferable because the variation in manufacturing is small even if the distance between the metal bodies 305 is 15 nm or less. As such an insulating film, silicon dioxide or silicon nitride used in a process for manufacturing a sidewall (side wall oxide film) of a semiconductor gate electrode is preferable. In this embodiment, a process using an insulating film is shown. However, it is only necessary to control the film thickness from the thin film formation (4) to the etching process (6), and a metal film may be used. For the etching used in these processes, RIE capable of fine processing is desirable.

(7) Formation of metal film, (8) Removal of partition plate The thickness of the metal film controls the height of the metal body 305. The thickness that effectively generates localized surface plasmons varies depending on the excitation wavelength used during measurement. A desirable thickness is 1000 nm or less. As a thin film forming method, vapor deposition, sputtering, CVD, PVD, or the like can be used. By using a general method for forming an alloy thin film, an alloy can be used as a material for the metal film. For example, sputtering using an alloy as a target can be used. Moreover, a plurality of types of metal thin films can be stacked by continuously performing a thin film forming method using a plurality of metal materials. For removing the partition plate, general wet (or dry) etching is performed. Specifically, hydrofluoric acid or a solution containing hydrofluoric acid is used together with silicon dioxide and silicon nitride.

(9) Resist application, (10) Patterning The size and shape of patterning are greatly related to the effect of localized surface plasmons. If the shape is similar to a triangle as shown in FIG. 3, one side of the triangle is preferably 1000 nm or less. As the resist 313, an electron beam negative positive can be used. Specifically, TEBN-1 (made by Tokuyama Co., Ltd.) is mentioned. After applying the resist with a spinner, it is pre-baked with a hot plate for about 2 to 5 minutes. After drawing with an electron beam having an acceleration voltage of 50 to 100 KV, development is performed with ethyl lactate, isopropanol, or ethanol.

(11) Etching, (12) Resist removal The metal body 305 is formed using the patterned resist as a mask. In order to increase pattern accuracy, RIE capable of fine processing is desirable. For the resist removal, a widely and commonly used ozone ashing process can be used.

(13) Probe immobilization When the probe 101 is a nucleic acid, various immobilization methods can be considered. As an example, a method using aminosilane treatment will be described. Aminosilane treatment is performed on the oxide film of the second metal 307 to introduce amino groups. Then, after reacting biotin-succinimide (NHS-Biotin manufactured by Pierce), streptavidin is bound. Next, a reaction site in which a probe is fixed between two adjacent metal bodies 305 can be completed by binding a probe whose end is modified with biotin in advance. Even if the probe 101 is a protein such as a nucleic acid synthase, it can be immobilized in the same manner. Specifically, N- (4-Maleimidobutyryloxy) succinimide (GMBS, manufactured by Dojindo Laboratories, Inc.) is reacted on the aminated oxide film and then reacted with nucleic acid synthase. Thus, the nucleic acid synthase can be immobilized. In addition, regardless of whether the probe 101 is a nucleic acid or a protein, a method using physical adsorption with nitrocellulose or polyacrylamide introduced on an oxide film, or a specific affinity between histidine and nickel ions or cobalt ions is used. For example, a method using biotin and avidin binding or a method using specific affinity between other proteins and peptides can be used.

  Next, a method for manufacturing a nucleic acid analysis device in which a probe is immobilized on an insulator will be described with reference to FIG. 4 with a focus on differences from FIG.

(1) Formation of Insulating Film After the insulating film 414 is formed on the support base 404 by vapor deposition, sputtering, CVD, PVD, etc., a metal film 407 is formed. The metal film 407 improves the adhesion between the insulating film 414 and the metal body 405. As the insulating film 414, an interlayer insulating film (manufactured by Hitachi Chemical Co., Ltd., HSG) that can be formed by a spin coater may be used. The process of (2) silicon film formation to (8) partition plate removal is the same as in FIG.

(A) Etching of Metal Film The metal film 407 is etched to expose the insulator 414 at the space 406 interface. Etching may be either dry (or wet) etching, but RIE capable of fine processing is desirable in order to improve processing accuracy. (9) Resist application to (13) The probe fixing process is performed in the same manner as in FIG.

  An example of a preferable configuration of the nucleic acid analysis device will be described with reference to FIG. A plurality of reaction regions 502 in which reaction sites including nanostructures are arranged in a lattice shape are mounted on the support base 501. As the reaction site, the structure in which the probe is fixed between two adjacent metal bodies described above is used. The arrangement interval is determined based on the nucleic acid sample to be analyzed and the specifications of the fluorescence detection apparatus. For example, if a reaction substrate 502 is 5 mm × 8 mm in which a slide glass of 25 mm × 75 mm is used as a support base 501 and reaction sites are arranged in a grid at intervals of 1 micrometer, 40 million kinds of nucleic acid molecules can be analyzed per region. About 8 regions can be mounted on the support base 501. Therefore, for example, when used for RNA expression analysis, since about 400,000 molecules of RNA are expressed per cell, RNA expression frequency analysis can be performed sufficiently accurately as in digital counting, About 8 analyzes can be performed on one substrate.

  As described above, the reaction on the device in which the plurality of reaction regions 502 are provided on the support substrate 501 is achieved by covering the reaction chamber 503 provided with the channel 504 in advance on the light-transmitting support substrate 501. it can. The reaction chamber 503 is made of a resin substrate such as PDMS (Polydimethylsiloxane) in which a groove of the flow path 504 is dug in advance, and is used by being bonded onto the device. More specifically, a temperature control unit 505 for storing / controlling the temperature of nucleic acid samples, reaction enzymes, buffers, nucleotide substrates, etc., a dispensing unit 506 for sending out the reaction liquid, a valve 507 for controlling the flow of the liquid, and a waste tank 508 Composed. If necessary, install a temperature controller to control the temperature. At the end of the reaction, the cleaning liquid is supplied through the flow path 504 of the reaction chamber 503 and stored in the waste liquid tank 508. Depending on the configuration of the flow path, the valve, and the dispensing unit, the reaction in each reaction region 502 can be performed sequentially or simultaneously.

  The size of the localized surface plasmon was calculated by simulation for the nucleic acid analysis device disclosed above. The material of the metal body 105 was ruthenium (Ru), rhodium (Rh), iridium (Ir), palladium (Pd), and ruthenium laminated on gold (Ru / Au). FIG. 6 shows the ratio between the intensity of irradiated light and the optical near-field intensity. Here, the optical near-field intensity was measured at the center of the space 106 of the support base 104. The calculation uses a finite element method (FEM), the wavelength of the excitation light 111 is 500 nm, the material of the support substrate 104 is quartz (refractive index: n = 1.45), and the surrounding medium 109 is water (refractive index: n = 1). .33). The optical near-field intensity necessary for obtaining a sufficient S / N ratio for detection is 45 or more, and it was found that a large localized surface plasmon can be obtained for each of the aforementioned substances.

An embodiment of the nucleic acid analyzer will be described with reference to FIG. In this embodiment, a means for supplying one or more types of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthase, primers, and nucleic acid samples to the nucleic acid analysis device, and light irradiation to the nucleic acid analysis device And fluorescence detection means for measuring the fluorescence of the fluorescent dye incorporated into the nucleic acid chain by the nucleic acid extension reaction caused by the coexistence of the nucleotide, the nucleic acid synthase, and the nucleic acid sample on the nucleic acid analysis device. More specifically, the device 705 is installed in a reaction chamber composed of a cover plate 701, a detection window 702, an inlet 703 that is a solution exchange port, and a discharge port 704. Note that PDMS (Polydimethylsiloxane) is used as the material of the cover plate 701 and the detection window 702. The thickness of the detection window 702 is 0.17 mm. Laser light 708 and 709 oscillated from a YAG laser light source (wavelength 532 nm, output 20 mW) 706 and a YAG laser light source (wavelength 355 nm, output 20 mW) 707 are reflected by the dichroic mirror 710 (reflects 410 nm or less). After adjusting so that it may become coaxial, it collects with the lens 711, and irradiates to the device 705 via a prism 712 above a critical angle. According to the present embodiment, localized surface plasmons are generated in the metal body existing on the surface of the device 705 by laser irradiation, and the target substance phosphor captured by the probe exists in the fluorescence enhancement field. . The phosphor is excited by laser light, and a part of the enhanced fluorescence is emitted through the detection window 702. The fluorescence emitted from the detection window 702 is converted into a parallel light beam by the objective lens 713 (× 60, NA 1.35, working distance 0.15 mm), and background light and excitation light are blocked by the optical filter 714, thereby forming an image. An image is formed on the two-dimensional CCD camera 716 by the lens 715.

  In the case of the sequential reaction method, as a nucleotide with a fluorescent dye, 3 at the 3′OH position of ribose as disclosed in “PNAS 2006, vol. 103, pp 19635-19640 (Non-patent Document 6)”. A '-O-allyl group can be used as a protecting group, and it can be linked to a fluorescent dye via an allyl group at the 5-position of pyrimidine or at the 7-position of purine. Since the allyl group is cleaved by light irradiation or contact with palladium, the quenching of the dye and the control of the extension reaction can be achieved simultaneously.

  Furthermore, in this example, as disclosed in “P.N.A.S. 2008, vol. 105, pp 1176-1181 (Non-patent Document 7)”, it is also possible to measure the elongation reaction in real time. As described above, by assembling a nucleic acid analyzer using the nucleic acid analysis device of this example, the analysis time can be shortened and the device and the analysis apparatus can be simplified without introducing a washing step, and only the sequential reaction method is used. In addition, it becomes possible to measure the elongation reaction of the base in real time, and the throughput can be greatly improved as compared with the conventional technique.

101, 201, 301, 401 Probe 102 Fluorescent dye 103 Fluorescent dye 104, 204, 304, 404, 501 of unreacted substrate Support base 105, 205, 305, 405 Metal body 106, 206, 306 Localized surface plasmon is generated Space 107, 207, 307 Second metal 108 Nucleic acid 209 Low refractive index material 310 Silicon 311 Insulator 312 Partition plate 313 Resist 407 Metal film 414 Insulating film 502 Reaction region 503 Reaction chamber 504 Channel 505 Temperature control unit 506 Dispensing unit 507 Valve 508 Waste liquid tank 701 Cover plate 702 Detection window 703 Inlet 704 Outlet 705 Device 706 YAG laser light source (wavelength 532 nm, output 20 mW)
707 YAG laser light source (wavelength 355 nm, output 20 mW)
708, 709 Laser light 710 Dichroic mirror 711 Lens 712 Prism 713 Objective lens 714 Optical filter 715 Imaging lens 716 Two-dimensional CCD camera

Claims (13)

A support base having a metal body that generates localized surface plasmons by light irradiation;
A nucleic acid analysis device for arranging a nucleic acid in a sample in a space where the localized surface plasmon is generated, and analyzing the nucleic acid by fluorescence measurement,
A nucleic acid analysis device in which the metal body is made of ruthenium. A support base having a metal body that generates localized surface plasmons by light irradiation;
A nucleic acid analysis device for arranging a nucleic acid in a sample in a space where the localized surface plasmon is generated, and analyzing the nucleic acid by fluorescence measurement,
A nucleic acid analysis device in which the metal body is made of iridium. A support base having a metal body that generates localized surface plasmons by light irradiation;
A nucleic acid analysis device for arranging a nucleic acid in a sample in a space where the localized surface plasmon is generated, and analyzing the nucleic acid by fluorescence measurement,
A nucleic acid analysis device in which the metal body is composed of palladium. A support base having a metal body that generates localized surface plasmons by light irradiation;
A nucleic acid analysis device for arranging a nucleic acid in a sample in a space where the localized surface plasmon is generated, and analyzing the nucleic acid by fluorescence measurement,
A nucleic acid analysis device in which the metal body is composed of rhodium. A support base having a metal body that generates localized surface plasmons by light irradiation;
A nucleic acid analysis device for arranging a nucleic acid in a sample in a space where the localized surface plasmon is generated, and analyzing the nucleic acid by fluorescence measurement,
A nucleic acid analysis device in which the metal body is composed of an alloy composed of two or more of ruthenium, iridium, palladium, rhodium, and platinum. The nucleic acid analysis device according to any one of claims 1 to 5,
The nucleic acid analysis device, wherein the wavelength of the light irradiation for generating the localized surface plasmon is 300 to 700 nm. The nucleic acid analysis device according to any one of claims 1 to 5,
A plurality of the metal bodies are present facing each other on the support base,
Localized surface plasmons are generated by light irradiation in the space where the metal body faces,
There is a second metal different from the metal body between the space and the support base,
A nucleic acid analysis device, wherein a probe for analyzing the nucleic acid in a sample is disposed on the second metal. The nucleic acid analysis device according to any one of claims 1 to 5,
There are a plurality of metal bodies facing each other on the support substrate,
Localized surface plasmons are generated by light irradiation in the space where the metal body faces,
An insulator exists between the space and the support substrate;
A nucleic acid analysis device, wherein a probe for analyzing a nucleic acid in a sample is disposed on the insulator. The nucleic acid analysis device according to any one of claims 7 and 8,
The nucleic acid analysis device, wherein the probe is one or more macromolecules selected from nucleic acids or proteins. The nucleic acid analysis device according to any one of claims 7 to 9,
A nucleic acid analysis device, wherein the probe is a single molecule. The nucleic acid analysis device according to any one of claims 7 to 10,
The nucleic acid analysis device, wherein the plurality of metal bodies facing each other are arranged in an array on the support substrate. The nucleic acid analysis device according to any one of claims 7 to 11,
The nucleic acid analysis device, wherein an interval between the plurality of metal bodies is 15 nm or less. A nucleic acid analysis apparatus using the nucleic acid analysis device according to any one of claims 1 to 12,
Means for supplying one or more types of biomolecules consisting of nucleotides, nucleotides having fluorescent dyes, nucleic acid synthase, primers, and nucleic acid samples to a nucleic acid analysis device;
Means for irradiating the nucleic acid analysis device with light;
In the nucleic acid chain by the nucleic acid extension reaction caused by the coexistence of one or more kinds of biomolecules consisting of the nucleotide, the nucleotide having the fluorescent dye, the nucleic acid synthase, the primer, and the nucleic acid sample on the nucleic acid analysis device. A fluorescence detection means for measuring the fluorescence of the incorporated fluorescent dye,
A nucleic acid analyzer characterized by acquiring base sequence information of the nucleic acid sample.

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