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1. 发明授权

US09290804B2 Microparticle having single-molecule nucleic acid probe, method for producing same, method for analyzing nucleic acid using same, device for nucleic acid analysis, and apparatus for analyzing nucleic acid 有权
公开(公告)号：US09290804B2
公开(公告)日：2016-03-22
申请号：US13981983
申请日：2012-01-26
申请人： Toshiro Saito , Kazumichi Imai , Takayuki Obara , Eri Tarasawa
发明人： Toshiro Saito , Kazumichi Imai , Takayuki Obara , Eri Tarasawa
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6869 , C12Q1/6806 , C12Q1/6834 , C12Q1/6876 , C12Q2525/131 , C12Q2525/197 , C12Q2535/122 , C12Q2537/125 , C12Q2537/159 , C12Q2563/155
摘要： A microparticle having a probe molecule able to capture a specific nucleic acid group to be analyzed is used to extract only the specific nucleic acid group to be analyzed from a nucleic acid sample and the microparticle is thereafter directly immobilized on a smooth plate, whereby a device for nucleic acid analysis is rapidly prepared. Immobilizing a single capture probe molecule onto an individual microparticle in advance and forming, at regular positions on the smooth substrate, an adhesion pad on which a functional group that binds to the microparticle has been introduced makes it possible to readily and rapidly prepare the device for nucleic analysis, where the nucleic acid sample to be analyzed is arranged molecule by molecule in a lattice shape on the smooth substrate.

2. 发明申请

US20130309675A1 Microparticle having single-molecule nucleic acid probe, method for producing same, method for analyzing nucleic acid using same, device for nucleic acid analysis, and apparatus for analyzing nucleic acid 有权
标题翻译：具有单分子核酸探针的微粒，其制备方法，使用其的核酸分析方法，核酸分析装置和核酸分析装置
公开(公告)号：US20130309675A1
公开(公告)日：2013-11-21
申请号：US13981983
申请日：2012-01-26
申请人： Toshiro Saito , Kazumichi Imai , Takayuki Obara , Eri Tarasawa
发明人： Toshiro Saito , Kazumichi Imai , Takayuki Obara , Eri Tarasawa
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6806 , C12Q1/6834 , C12Q1/6876 , C12Q2525/131 , C12Q2525/197 , C12Q2535/122 , C12Q2537/125 , C12Q2537/159 , C12Q2563/155
摘要： A microparticle having a probe molecule able to capture a specific nucleic acid group to be analyzed is used to extract only the specific nucleic acid group to be analyzed from a nucleic acid sample and the microparticle is thereafter directly immobilized on a smooth plate, whereby a device for nucleic acid analysis is rapidly prepared. Immobilizing a single capture probe molecule onto an individual microparticle in advance and forming, at regular positions on the smooth substrate, an adhesion pad on which a functional group that binds to the microparticle has been introduced makes it possible to readily and rapidly prepare the device for nucleic analysis, where the nucleic acid sample to be analyzed is arranged molecule by molecule in a lattice shape on the smooth substrate.
摘要翻译：使用具有能够捕获要分析的特定核酸组的探针分子的微粒从核酸样品中仅提取待分析的特异性核酸组，然后将微粒直接固定在光滑平板上，用于核酸分析快速准备。将单个捕获探针分子预先固定在单个微粒上，并且在光滑基底上的规则位置处形成粘附有与微粒结合的官能团的粘合垫，使得可以容易且快速地制备用于核酸分析，其中要分析的核酸样品以平滑基底上的格子状分子排列。

3. 发明申请

US20130157264A1 NUCLEIC ACID ANALYSIS DEVICE, NUCLEIC ACID ANALYSIS APPARATUS, AND NUCLEIC ACID ANALYSIS METHOD 审中-公开
标题翻译：核酸分析装置，核酸分析装置和核酸分析方法
公开(公告)号：US20130157264A1
公开(公告)日：2013-06-20
申请号：US13818704
申请日：2011-07-19
申请人： Takayuki Obara , Kazumichi Imai , Toshiro Saito , Satoshi Takahashi
发明人： Takayuki Obara , Kazumichi Imai , Toshiro Saito , Satoshi Takahashi
IPC分类号： G01N21/64
CPC分类号： G01N21/6486 , C12Q1/6837 , G01N21/6428 , G01N21/6452 , G01N21/648 , C12Q2563/173
摘要： In a nucleic acid analysis device which detects a fluorescent dye on a nucleic acid sample immobilized on a surface of a substrate by exciting the fluorescent dye with an evanescent wave, the detection of a fluorescence signal with a high SN ratio is realized even for a long nucleic acid sample.The nucleic acid analysis device according to the invention is a nucleic acid analysis device in which a plurality of regions for immobilizing a nucleic acid sample are provided on a surface of a support base and a single molecule of a nucleic acid sample is immobilized on at least one of the regions, and which performs sequence determination by performing an extension reaction of the immobilized nucleic acid sample, wherein the immobilization of the single molecule of the nucleic acid sample on the support base is performed at two or more points.
摘要翻译：在通过用ev逝波激发荧光染料来检测固定在基板表面上的核酸样品上的荧光染料的核酸分析装置中，即使长时间地实现高SN比的荧光信号的检测核酸样品。根据本发明的核酸分析装置是核酸分析装置，其中在支持基底的表面上提供用于固定核酸样品的多个区域，并且将至少一个核酸样品的单个分子固定在至少其中一个区域，并且通过进行固定化的核酸样品的延伸反应进行序列测定，其中将核酸样品的单分子固定在载体基底上进行两点或更多点。

4. 发明申请

US20110281320A1 DEVICE FOR ANALYZING NUCLEIC ACIDS AND APPARATUS FOR ANALYZING NUCLEIC ACIDS 审中-公开
标题翻译：用于分析核酸的装置和分析核酸的装置
公开(公告)号：US20110281320A1
公开(公告)日：2011-11-17
申请号：US13147117
申请日：2010-01-18
申请人： Toshiro Saito , Kazumichi Imai
发明人： Toshiro Saito , Kazumichi Imai
IPC分类号： C12N11/14 , C12M1/34 , C07H21/00 , C12M1/40 , C12N11/00
CPC分类号： C12Q1/6816 , C12Q2565/50 , C12Q2563/143
摘要： An object of the present invention is to regularly align microparticles, on each of which a nucleic acid synthetase or a DNA probe capable of capturing a nucleic acid sample fragment is immobilized, on a support so as to improve throughput of nucleic acid analysis. The present invention relates to a method comprising immobilizing a nucleic acid synthetase, a DNA probe, or the like in advance to a microparticle, forming a pattern of metal pads each having a diameter smaller than the microparticle diameter with gold or the like on a support, and allowing a microparticle to be bound to the pads via a chemical bond. In addition, when the surfaces of microparticles are electrically charged, a pattern of metal pads each having a diameter equivalent to or larger than the microparticle diameter is formed with gold or the like on a support and a microparticle is allowed to be bound to the pads via a chemical bond. According to the present invention, many types of nucleic acid fragment samples can be regularly aligned at a high density and immobilized on a support. This allows high throughput analysis of nucleic acid samples. For example, if microparticles are immobilized at 1-micron pitches, a high density of 106 nucleic acid fragments/emm2 can be readily achieved.
摘要翻译：本发明的一个目的是在载体上规则地将微粒（其中每一个核酸合成酶或能够捕获核酸样品片段的DNA探针）定位在载体上，以便提高核酸分析的生产能力。本发明涉及一种将核酸合成酶，DNA探针等预先固定在微粒上的方法，在支撑体上形成具有小于微粒径的直径的金属垫的图案，并且允许微粒通过化学键与焊盘结合。此外，当微粒表面带电时，在支撑体上形成金等等于或大于微粒直径的金属垫的图案，并且允许微粒结合到垫通过化学键。根据本发明，许多类型的核酸片段样品可以高密度地定期排列并固定在载体上。这允许核酸样品的高通量分析。例如，如果微粒以1微米间距固定，则可以容易地实现高密度的106个核酸片段/ emm2。

5. 发明授权

US09207235B2 Nucleic acid analyzer, reaction device for nucleic acid analysis and substrate of reaction device for nucleic acid analysis 有权
标题翻译：核酸分析仪，核酸分析反应装置和核酸分析反应装置底物
公开(公告)号：US09207235B2
公开(公告)日：2015-12-08
申请号：US13575564
申请日：2010-12-01
申请人： Yoshiaki Sugimura , Masatoshi Narahara , Kazumichi Imai , Toshiro Saito , Ryoji Inaba , Takuya Matsui
发明人： Yoshiaki Sugimura , Masatoshi Narahara , Kazumichi Imai , Toshiro Saito , Ryoji Inaba , Takuya Matsui
IPC分类号： B01L3/00 , G01N33/543 , G01N21/64 , G01N33/53 , G01N33/58 , G01N35/00
CPC分类号： G01N33/54313 , B01L3/502715 , B01L2200/0668 , B01L2300/0609 , B01L2300/0877 , B01L2300/0883 , G01N21/6452 , G01N21/648 , G01N33/5308 , G01N33/54373 , G01N33/582 , G01N35/00 , G01N2035/00564
摘要： Provided is a reaction device for nucleic acid analysis wherein microparticles, which carry a nucleic acid to be detected having been immobilized thereon, are aligned in a lattice form on a substrate according to the pixel size of a two-dimensional sensor. By this reaction device for nucleic acid analysis which is provided with a channel-forming reaction chamber on the substrate (101), the nucleic acid having been immobilized on the microparticles (103) on the substrate (101) is detected. The microparticles (103), which carry the nucleic acid to be detected having been immobilized thereon, are arranged by microstructures (102) aligned on the substrate (101).
摘要翻译：提供了用于核酸分析的反应装置，其中携带已被固定在其上的被检测核酸的微粒根据二维传感器的像素尺寸以格子形式排列在基板上。通过在基板（101）上设置有通道形成反应室的核酸分析用反应装置，检测固定在基板（101）上的微粒（103）上的核酸。携带已被固定在其上的被检测核酸的微粒（103）通过在基板（101）上排列的微结构（102）布置。

6. 发明申请

US20140200162A1 METHOD AND APPARATUS FOR NUCLEIC ACID ANALYSIS 审中-公开
标题翻译：核酸分析方法与装置
公开(公告)号：US20140200162A1
公开(公告)日：2014-07-17
申请号：US14233256
申请日：2012-05-16
申请人： Toshiro Saito , Koshin Hamasaki , Satoshi Takahashi , Muneo Maeshima , Kyoko Imai , Kazumichi Imai , Ryuji Tao
发明人： Toshiro Saito , Koshin Hamasaki , Satoshi Takahashi , Muneo Maeshima , Kyoko Imai , Kazumichi Imai , Ryuji Tao
IPC分类号： C12Q1/68 , C12N15/10
CPC分类号： C12Q1/6834 , C12N15/1065 , C12Q2525/207 , C12Q2537/125 , C12Q2537/143 , C12Q2563/143 , C12Q2563/149
摘要： A convenient method for nucleic acid analysis is provided, which enables 1000 or more types of nucleic acid to be analyzed collectively with high comprehensiveness and with a dynamic range of at least four digits. In particular, provided is a very effective analytical method especially for untranslated RNAs and microRNAs, of which the types of target nucleic acids is 10000 or lower. Nucleic acids can be analyzed conveniently and rapidly with high comprehensiveness and quantitative performance at single-molecule sensitivity and resolution by following the steps of: preparing a group of target nucleic acid fragments one molecule at a time and hybridizing the nucleic acid molecules, which have known base sequences and have been labeled with the fluorescence substances, with the group of the target nucleic acid fragments to detect the fluorescence substances labeling the hybridized nucleic acid molecules.
摘要翻译：提供了一种方便的核酸分析方法，使得能够以高全面性和至少四位数的动态范围共同分析1000种或更多种类型的核酸。特别地，提供了非常有效的分析方法，特别是对于非翻译的RNA和微小RNA，其靶核酸的类型为10000或更低。可以通过以下步骤以单分子灵敏度和分辨率的高全面性和定量性能方便快速地分析核酸：一次一个分子制备一组靶核酸片段，并将已知的核酸分子杂交碱基序列，并用荧光物质标记，用靶核酸片段组来检测标记杂交核酸分子的荧光物质。

7. 发明授权

US10294519B2 Method and apparatus for nucleic acid analysis 有权
公开(公告)号：US10294519B2
公开(公告)日：2019-05-21
申请号：US14233256
申请日：2012-05-16
申请人： Toshiro Saito , Koshin Hamasaki , Satoshi Takahashi , Muneo Maeshima , Kyoko Imai , Kazumichi Imai , Ryuji Tao
发明人： Toshiro Saito , Koshin Hamasaki , Satoshi Takahashi , Muneo Maeshima , Kyoko Imai , Kazumichi Imai , Ryuji Tao
IPC分类号： C12Q1/6834 , C12N15/10
摘要： A convenient method for nucleic acid analysis is provided, which enables 1000 or more types of nucleic acid to be analyzed collectively with high comprehensiveness and with a dynamic range of at least four digits. In particular, provided is a very effective analytical method especially for untranslated RNAs and microRNAs, of which the types of target nucleic acids is 10000 or lower. Nucleic acids can be analyzed conveniently and rapidly with high comprehensiveness and quantitative performance at single-molecule sensitivity and resolution by following the steps of: preparing a group of target nucleic acid fragments one molecule at a time and hybridizing the nucleic acid molecules, which have known base sequences and have been labeled with the fluorescence substances, with the group of the target nucleic acid fragments to detect the fluorescence substances labeling the hybridized nucleic acid molecules.

8. 发明申请

US20120316087A1 Nucleic Acid Analyzer, Reaction Device for Nucleic Acid Analysis and Substrate of Reaction Device for Nucleic Acid Analysis 有权
标题翻译：核酸分析仪，用于核酸分析的反应装置和用于核酸分析的反应装置的底物
公开(公告)号：US20120316087A1
公开(公告)日：2012-12-13
申请号：US13575564
申请日：2010-12-01
申请人： Yoshiaki Sugimura , Masatoshi Narahara , Kazumichi Imai , Toshiro Saito , Ryoji Inaba , Takuya Matsui
发明人： Yoshiaki Sugimura , Masatoshi Narahara , Kazumichi Imai , Toshiro Saito , Ryoji Inaba , Takuya Matsui
IPC分类号： C40B60/10
CPC分类号： G01N33/54313 , B01L3/502715 , B01L2200/0668 , B01L2300/0609 , B01L2300/0877 , B01L2300/0883 , G01N21/6452 , G01N21/648 , G01N33/5308 , G01N33/54373 , G01N33/582 , G01N35/00 , G01N2035/00564
摘要： Provided is a reaction device for nucleic acid analysis wherein microparticles, which carry a nucleic acid to be detected having been immobilized thereon, are aligned in a lattice form on a substrate according to the pixel size of a two-dimensional sensor. By this reaction device for nucleic acid analysis which is provided with a channel-forming reaction chamber on the substrate (101), the nucleic acid having been immobilized on the microparticles (103) on the substrate (101) is detected. The microparticles (103), which carry the nucleic acid to be detected having been immobilized thereon, are arranged by microstructures (102) aligned on the substrate (101).
摘要翻译：提供了用于核酸分析的反应装置，其中携带已被固定在其上的被检测核酸的微粒根据二维传感器的像素尺寸以格子形式排列在基板上。通过在基板（101）上设置有通道形成反应室的核酸分析用反应装置，检测固定在基板（101）上的微粒（103）上的核酸。携带已被固定在其上的被检测核酸的微粒（103）通过在基板（101）上排列的微结构（102）布置。

9. 发明授权

US08073219B2 Nucleic acid analyzing apparatus 有权
标题翻译：核酸分析仪
公开(公告)号：US08073219B2
公开(公告)日：2011-12-06
申请号：US12369342
申请日：2009-02-11
申请人： Akira Maekawa , Toshiro Saito , Kiyoyuki Kagii , Takayuki Obara
发明人： Akira Maekawa , Toshiro Saito , Kiyoyuki Kagii , Takayuki Obara
IPC分类号： G06K9/18
CPC分类号： G01N21/6452 , G01N21/648 , G06T7/0004 , G06T7/136 , G06T2207/10064 , G06T2207/30024
摘要： The present invention provides a nucleic acid analyzing apparatus which achieves highly accurate analytical ability even in single molecule DNA analysis. The nucleic acid analyzing apparatus detects locations of fluorescent bright spots in image information about light emission, deletes defective bright spots, and thereby creates intensity trace data about proper bright spots.
摘要翻译：本发明提供即使在单分子DNA分析中也能够获得高精度的分析能力的核酸分析装置。核酸分析装置检测关于发光的图像信息中的荧光亮点的位置，删除有缺陷的亮点，从而生成关于适当的亮点的强度追踪数据。

10. 发明授权

US09957562B2 Nucleic acid analysis device and nucleic acid analyzer 有权
公开(公告)号：US09957562B2
公开(公告)日：2018-05-01
申请号：US14002125
申请日：2012-01-24
申请人： Koshin Hamasaki , Toshiro Saito , Takayuki Obara
发明人： Koshin Hamasaki , Toshiro Saito , Takayuki Obara
IPC分类号： C12Q1/68 , G01N21/64 , C40B40/06
CPC分类号： C12Q1/6876 , C12Q1/6834 , C12Q1/6874 , C40B40/06 , G01N21/6452 , G01N21/648 , G01N2021/6439 , C12Q2521/543 , C12Q2535/122 , C12Q2563/149 , C12Q2565/513 , C12Q2523/308
摘要： In the conventional nucleic acid analysis devices and nucleic acid analyzers, there was no technique available for sequencing a single nucleic acid molecule easily and highly efficiently. The present invention enabled a highly efficient single molecule immobilization of nucleic acid with good reproductivity in a short time at a low price by providing small metallic bonding pads at predetermined positions on a support substrate, firmly fixing a hydrophobic linker on the bonding pads, and bonding on to the linker bulky microparticles onto which a single molecule of a nucleic acid sample fragment is immobilized. According to the present invention, in the nucleic acid analysis device which uses a nucleic acid analyzer, the nucleotide read length can be extended and many types of nucleic acid molecule to be analyzed can be analyzed at one time.

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