JPH0688779A - Gene sensor - Google Patents
Gene sensorInfo
- Publication number
- JPH0688779A JPH0688779A JP4238607A JP23860792A JPH0688779A JP H0688779 A JPH0688779 A JP H0688779A JP 4238607 A JP4238607 A JP 4238607A JP 23860792 A JP23860792 A JP 23860792A JP H0688779 A JPH0688779 A JP H0688779A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- sensor
- nucleotide
- electrode
- chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 83
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 56
- 239000002773 nucleotide Substances 0.000 claims abstract description 47
- 239000000126 substance Substances 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000001514 detection method Methods 0.000 claims description 35
- 230000000295 complement effect Effects 0.000 claims description 10
- 239000013078 crystal Substances 0.000 abstract description 25
- 230000010355 oscillation Effects 0.000 abstract description 17
- 239000004793 Polystyrene Substances 0.000 abstract description 12
- 229920002223 polystyrene Polymers 0.000 abstract description 12
- 239000007788 liquid Substances 0.000 abstract description 5
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 230000004048 modification Effects 0.000 abstract 1
- 238000012986 modification Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 29
- 238000000034 method Methods 0.000 description 17
- 230000008859 change Effects 0.000 description 12
- 239000007822 coupling agent Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 6
- 239000010931 gold Substances 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 229910052737 gold Inorganic materials 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 239000003607 modifier Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000010419 fine particle Substances 0.000 description 4
- -1 methylol groups Chemical group 0.000 description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 239000006087 Silane Coupling Agent Substances 0.000 description 3
- 239000002156 adsorbate Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000010897 surface acoustic wave method Methods 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000010408 film Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229910000859 α-Fe Inorganic materials 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910001922 gold oxide Inorganic materials 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、遺伝子検査および遺伝
子診断に用いられるバイオセンサーに関する。詳しく
は、簡易操作可能で迅速計測に使用する遺伝子センサー
に関する。TECHNICAL FIELD The present invention relates to a biosensor used for genetic testing and genetic diagnosis. Specifically, it relates to a gene sensor that can be easily operated and is used for rapid measurement.
【０００２】[0002]
【従来の技術】従来、遺伝子検査および遺伝子診断に
は、ドット ブロット（Dot blot）法、イン シテュ
ハイブリダイゼーション（in situ Hybridization）
法、サザンブロッティング（Southern blotting）法、
ＰＣＲ法、多形ＤＮＡマーカー法などが知られている。
ドット ブロット法およびイン シテュ ハイブリダイ
ゼーション法は、測定操作が簡単で操作時間が比較的短
いという特長があるが、検出感度が低い。サザン ブロ
ッティング法は、検出感度が高いが、操作が煩雑で長時
間かかる。十分な感度を得るためにはＰＣＲ法、ＬＣＲ
法により被検査部位のＤＮＡを１０万〜１００万倍に増
幅することがなされている。ＰＣＲ法は極めて感度が高
いが、操作条件が複雑であるうえ、コンタミネーション
の虞れもある。また、何れの方法も検出にはアイソトー
プを用いるため管理区域を設ける必要があり、取り扱い
が煩雑で、集中管理された中央検査部での一括検査から
医療現場への、即時対応を目的とした分散化の流れにも
対応できない。また、廃棄処理の問題もある。2. Description of the Related Art Conventionally, a dot blot method and an in situ method have been used for genetic testing and diagnostics.
Hybridization (in situ Hybridization)
Method, Southern blotting method,
PCR method, polymorphic DNA marker method and the like are known.
The dot blot method and in situ hybridization method have the characteristics that the measurement operation is simple and the operation time is relatively short, but the detection sensitivity is low. The Southern blotting method has high detection sensitivity, but the operation is complicated and takes a long time. PCR method and LCR to obtain sufficient sensitivity
According to the method, the DNA at the site to be inspected is amplified 100,000 to 1,000,000 times. The PCR method has extremely high sensitivity, but the operating conditions are complicated, and there is a risk of contamination. In addition, since all methods use an isotope for detection, it is necessary to set up a control area, and handling is complicated, and it is distributed for the purpose of immediate response from collective inspection at centrally controlled centralized inspection department to medical field. It cannot cope with the flow of change. There is also the problem of disposal.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、このよ
うな従来の放射線標識ＤＮＡ検査装置にあっては、時間
がかかる、放射線のため一般検査室では使用できないと
いった問題があった。このために、特に高感度でアイソ
トープを用いない遺伝子センサーの開発が渇望されてい
る。However, such a conventional radiolabeled DNA inspection apparatus has problems that it takes time and cannot be used in a general inspection room due to radiation. For this reason, there is a strong demand for the development of a gene sensor that is particularly sensitive and does not use isotopes.
【０００４】本発明は、このような従来のアイソトープ
を用いるＤＮＡセンサーの検出原理に関する問題点と、
高感度簡易迅速測定センサーがないという問題点に鑑み
てなされたものである。すなわち、修飾ヌクレオチド鎖
を固定化し、超高感度化した電気機械変換素子からなる
遺伝子センサーを提供することを目的とする。The present invention has the problems relating to the detection principle of a DNA sensor using such a conventional isotope,
This has been made in view of the problem that there is no highly sensitive simple quick measurement sensor. That is, it is an object of the present invention to provide a gene sensor comprising an electromechanical conversion element having a modified nucleotide chain immobilized thereon and having an extremely high sensitivity.
【０００５】[0005]
【課題を解決するための手段】上記課題は、電気機械変
換素子と、少なくとも一部が検出目的遺伝子と相補的塩
基配列を有する該電気機械変換素子の電極表面に固定さ
れた第１のヌクレオチド鎖と、該第１のヌクレオチド鎖
と相補結合している第２のヌクレオチド鎖とを有する遺
伝子センサーであって、該第２のヌクレオチド鎖には検
出目的遺伝子よりも大きな質量をもつ物質が固定されて
なる遺伝子センサ−によって解決される。[Means for Solving the Problems] The above object is to provide an electromechanical conversion element and a first nucleotide chain immobilized on the electrode surface of the electromechanical conversion element, at least a part of which has a base sequence complementary to the gene of interest. And a second nucleotide chain that is complementary bonded to the first nucleotide chain, wherein a substance having a mass larger than that of the detection target gene is immobilized on the second nucleotide chain. It is solved by the gene sensor.
【０００６】また、上記電気機械変換素子が電気音響ト
ランスデューサーである遺伝子センサーによっても解決
される。また、上記電気機械変換素子が表面弾性波素子
である遺伝子センサーによっても解決される。また、上
記電気機械変換素子がバルク弾性波素子である遺伝子セ
ンサーによっても解決される。また、上記電気機械変換
素子が水晶発振子である遺伝子センサーによっても解決
される。なお、本発明において電気機械変換素子とは電
気−機械変換機能、あるいは電気−機械−光変換機能を
持つ素子である。The electromechanical conversion element can be solved by a gene sensor which is an electroacoustic transducer. Further, the electromechanical conversion element is also solved by a gene sensor in which the surface acoustic wave element is used. Further, the electromechanical conversion element is also solved by a gene sensor in which a bulk acoustic wave element is used. Further, the electromechanical conversion element is also solved by a gene sensor in which the crystal oscillator is used. In the present invention, the electromechanical conversion element is an element having an electromechanical conversion function or an electromechanical light conversion function.
【０００７】また、上記物質の質量が０.０１ｐｇ〜１
ｎｇである遺伝子センサーによっても解決される。ま
た、上記物質の比重が１ｇ／ｃｍ3以上である遺伝子セ
ンサーによっても解決される。また、上記物質の直径が
0.01〜10μｍである遺伝子センサーによっても解決され
る。また、上記物質の材質が合成樹脂、磁性体、金属ま
たは金属酸化物のいずれかである遺伝子センサーによっ
ても解決される。また、上記物質の材質がポリスチレン
またはテフロンである遺伝子センサーによっても解決さ
れる。また、上記物質の材質が金または酸化チタンであ
る遺伝子センサーによっても解決される。また、上記物
質の材質がフェライトである遺伝子センサーによっても
解決される。The mass of the above substance is 0.01 pg-1.
It is also solved by the gene sensor which is ng. It can also be solved by a gene sensor in which the specific gravity of the above substance is 1 g / cm 3 or more. Also, if the diameter of the substance is
It is also solved by a gene sensor that is 0.01-10 μm. Further, the problem can be solved also by a gene sensor in which the material of the above substance is a synthetic resin, a magnetic substance, a metal or a metal oxide. Further, the problem can also be solved by a gene sensor in which the material of the above substance is polystyrene or Teflon. Further, the problem can be solved by a gene sensor in which the material of the above substance is gold or titanium oxide. Also, the problem can be solved by a gene sensor in which the material of the above substance is ferrite.
【０００８】さらに、上記物質が、タンパク質や検出目
的遺伝子以外の核酸等の非特異的吸着や粒子自体の凝集
が起こらないような吸着性の低い材質からなる遺伝子セ
ンサーによっても解決される。Further, the above-mentioned substance can be solved by a gene sensor made of a material having low adsorptivity so that non-specific adsorption of proteins and nucleic acids other than the detection target gene and aggregation of particles themselves do not occur.
【０００９】[0009]
【作用】本発明の遺伝子センサーは、検出目的の遺伝子
が該遺伝子と同配列を持つセンサーに結合された被修飾
ヌクレオチド鎖と相同交換するときに生ずる質量変化の
差を電気機械変換素子の振動の変化として高感度に計測
する。すなわち、置換される被修飾ヌクレオチド鎖に結
合した修飾物質の質量を検出目的遺伝子のそれより大き
く、好ましくは0.1〜１０ｐｇとしておくことで高感度
の遺伝子の検出同定が迅速に行うことができる。測定時
には遺伝子の相同置換の速度を速くするため、検出目的
遺伝子を含む被検液の温度を水の沸点まで上げてもよ
い。In the gene sensor of the present invention, the difference in mass change caused when the gene to be detected undergoes homologous exchange with the modified nucleotide chain bound to the sensor having the same sequence as the gene causes the difference in vibration of the electromechanical transducer. The change is measured with high sensitivity. That is, by setting the mass of the modified substance bound to the modified nucleotide chain to be replaced to be larger than that of the detection target gene, preferably 0.1 to 10 pg, the highly sensitive detection and identification of the gene can be carried out rapidly. At the time of measurement, the temperature of the test liquid containing the gene of interest may be raised to the boiling point of water in order to accelerate the rate of homologous substitution of the gene.
【００１０】以下に本発明を詳細に説明する。電気機械
変換素子の電極表面に固定化されているヌクレオチド鎖
に相補結合している被修飾ヌクレオチド鎖が検出目的遺
伝子と相同交換すると、被修飾ヌクレオチド鎖に結合し
ている修飾物質も被修飾ヌクレオチド鎖とともに電極表
面から外れる。このとき［検出目的遺伝子−修飾物質−
被修飾ヌクレオチド鎖］だけの質量変化（＝ｘ）が生じ
る。修飾物質がない場合、［検出目的遺伝子−被修飾ヌ
クレオチド鎖］だけの質量変化（＝ｙ）となる。ここ
で、修飾物質の質量を検出目的遺伝子のそれより大きく
すれば化学変化（遺伝子相同交換反応）を引き金にして
質量変化を（ｘ／ｙ）倍増幅したことになる。The present invention will be described in detail below. When the modified nucleotide chain complementary to the nucleotide chain immobilized on the electrode surface of the electromechanical conversion element is homologously exchanged with the target gene for detection, the modified substance bound to the modified nucleotide chain also includes the modified nucleotide chain. Together with it, it comes off from the electrode surface. At this time, [detection target gene-modifying substance-
Mass change (= x) of only the modified nucleotide chain] occurs. When there is no modifier, the mass change (= y) of [target gene-modified nucleotide chain] only occurs. Here, if the mass of the modifying substance is made larger than that of the detection target gene, it means that the chemical change (gene homologous exchange reaction) is triggered to amplify the mass change by (x / y) times.
【００１１】検出目的遺伝子をヒト遺伝子のような巨大
ＤＮＡとし、分子量１０9と仮定しても、１分子当たり
の質量は（１０9／6.02×１０23＝）１.７×１０-15ｇ
である。一方、修飾物質に直径約１.１μｍのポリスチ
レン粒子を用いると、その質量は１ｐｇ以上になるの
で、質量変化は１０００倍以上にも増幅される。Even if the target gene for detection is a huge DNA such as a human gene and the molecular weight is 10 9 , the mass per molecule is (10 9 /6.02×10 23 =) 1.7 × 10 -15 g
Is. On the other hand, when polystyrene particles having a diameter of about 1.1 μm are used as the modifying substance, the mass thereof is 1 pg or more, so the mass change is amplified 1000 times or more.
【００１２】検出目的遺伝子を含む被検液中に存在する
検出目的遺伝子以外の核酸類やタンパク質類等の電極表
面への非特異的吸着によって質量の増加が起こる。従っ
て、修飾物質が結合したヌクレオチド鎖を用いない、す
なわち質量増加方式の遺伝子センサーでは、非特異的吸
着の質量の変化方向と同方向となるので検出目的遺伝子
と非特異的吸着物との選択性に劣るものとなる。これに
対し、本発明の遺伝子センサーは非特異的吸着物が電極
表面に吸着しても、検出目的遺伝子の結合による質量の
変化は減少方向であり非特異的吸着物とは逆であるので
選択性に優れた遺伝子センサーとなっている。An increase in mass occurs due to nonspecific adsorption of nucleic acids and proteins other than the detection target gene existing in the test liquid containing the detection target gene onto the electrode surface. Therefore, in the case of a mass-increasing gene sensor that does not use a nucleotide chain to which a modifier is bound, the direction of change in the mass of nonspecific adsorption is in the same direction, so the selectivity between the target gene of detection and the nonspecific adsorbate is high. Will be inferior to. On the other hand, the gene sensor of the present invention is selected because the change in mass due to the binding of the target gene for detection is in the decreasing direction even when the nonspecific adsorbate is adsorbed on the electrode surface, which is the opposite of the nonspecific adsorbate It is a gene sensor with excellent properties.
【００１３】以下、本発明を実施態様に基づいてより詳
細に説明する。本発明の遺伝子センサーに用いる電気機
械変換素子としては一般に広く用いられているものを適
宜利用することができる。すなわち、電気−機械変換機
能あるいは電気−機械−光変換機能を持つ素子であれば
良く、表面弾性波素子あるいはバルク弾性波素子のいず
れであっても使用することができる。表面弾性波素子と
しては、ＳＡＷデバイス等が挙げられ、バルク弾性波素
子としては、水晶発振子等が挙げられる。尚、ここでは
水晶発振子を例にとって図面を参照しつつ説明を続け
る。Hereinafter, the present invention will be described in more detail based on embodiments. As the electromechanical conversion element used for the gene sensor of the present invention, those generally widely used can be appropriately used. That is, any element having an electro-mechanical conversion function or an electro-mechanical-optical conversion function may be used, and either a surface acoustic wave element or a bulk acoustic wave element can be used. The surface acoustic wave element may be a SAW device or the like, and the bulk acoustic wave element may be a crystal oscillator or the like. Note that the description will be continued here with reference to the drawings using a crystal oscillator as an example.
【００１４】第１図は本発明にかかる遺伝子センサー１
０の模式図である。水晶発振子１は水晶板２とその側面
の電極３とを有する。この電極３の表面に検出目的遺伝
子の少なくとも一部と相補的な塩基配列を有するヌクレ
オチド鎖５を固定化する。このときヌクレオチド鎖５と
相補的なヌクレオチド鎖５'（後で修飾物質７と結合す
る）とは相補結合してヌクレオチド対６を形成してい
る。ヌクレオチド鎖５を電極表面に固定化する際には
３'−末端、５'−末端、あるいは本発明の目的を阻害し
ない他の箇所の１箇所ないし数箇所で結合させることが
できるが、この部分での結合手段、あるいは後述するヌ
クレオチド鎖５'と修飾物質７との結合手段は本発明の
主たる要素ではなく、公知のあるいは今後生み出される
種々の手段を適用することができるが、ここでは操作の
容易な５'−末端を固定する態様に基づいて説明する。FIG. 1 shows a gene sensor 1 according to the present invention.
It is a schematic diagram of 0. The crystal oscillator 1 has a crystal plate 2 and electrodes 3 on its side surface. On the surface of this electrode 3, a nucleotide chain 5 having a base sequence complementary to at least a part of the detection target gene is immobilized. At this time, the nucleotide chain 5 and the complementary nucleotide chain 5 ′ (which will be bonded to the modifier 7 later) are complementary-bonded to each other to form a nucleotide pair 6. When the nucleotide chain 5 is immobilized on the surface of the electrode, it can be bound at one or several positions of the 3'-end, the 5'-end, or another position that does not impair the object of the present invention. The binding means in step 1, or the binding means between the nucleotide chain 5 ′ and the modifying substance 7 described later is not the main element of the present invention, and various known or later-developed means can be applied. An explanation will be given based on the mode of fixing the easy 5'-end.
【００１５】まず、電極３の表面にヌクレオチド鎖５の
５'−末端のリン酸基と反応性を有し、電極３にヌクレ
オチド鎖５を固定可能とするカップリング剤４を被覆す
る。カップリング剤４の具体例としてはシランカップリ
ング剤等が挙げられる。このカップリング剤の被覆はセ
ンサーの使用中に溶け出してヌクレトチド鎖５が遺伝子
センサー１０から外れてしまわないようなものでなけれ
ばならないが、例えば、カップリング剤をトルエン等
の溶媒で希釈液とし、これを電極３の表面に塗布し、乾
燥する方法、真空チャンバー内にカップリング剤の有
機溶媒溶液と水晶発振子とを入れ、減圧下で２時間ほど
吸着させた後、常圧下に戻してから乾燥させる方法、
スピンコーター装置を用いてカップリング剤薄膜を形成
した後、乾燥させる方法、がある。First, the surface of the electrode 3 is coated with a coupling agent 4 which is reactive with the phosphate group at the 5'-end of the nucleotide chain 5 and can fix the nucleotide chain 5 on the electrode 3. Specific examples of the coupling agent 4 include silane coupling agents and the like. The coating of this coupling agent must be such that it will not dissolve out during use of the sensor and the nucleotide chain 5 will not come off from the gene sensor 10. For example, the coupling agent should be diluted with a solvent such as toluene. , A method of applying this to the surface of the electrode 3 and drying it, putting an organic solvent solution of a coupling agent and a crystal oscillator in a vacuum chamber, adsorbing for 2 hours under reduced pressure, and then returning to normal pressure. How to dry from,
There is a method in which a coupling agent thin film is formed using a spin coater device and then dried.
【００１６】尚、理論上はカップリング剤４の電極３表
面における存在量は固定化されるヌクレオチド鎖５と同
じだけあればよいのではあるが、実際には単分子膜〜数
分子層が好ましい。これ以上の膜厚となるとヌクレオチ
ド鎖５のないサイトが多くなり、非特異物質の吸着影響
が大きくなるので好ましくない。In theory, the amount of the coupling agent 4 present on the surface of the electrode 3 should be the same as that of the immobilized nucleotide chain 5, but in practice, a monomolecular film to several molecular layers are preferable. . If the film thickness is larger than this, the number of sites without the nucleotide chain 5 increases and the influence of adsorption of non-specific substances increases, which is not preferable.
【００１７】このカップリング剤４が被覆された水晶発
振子１の電極３表面に、さらにヌクレオチド対６を結合
する。すなわち、ヌクレオチド対６を含む水溶液に水晶
発振子１を浸漬し、カルボジイミド存在下でヌクレオチ
ド鎖５の５'−末端のリン酸基とカップリング剤のＯＨ
基とを結合して、ヌクレオチド鎖５を電極３表面に固定
した。A nucleotide pair 6 is further bonded to the surface of the electrode 3 of the crystal oscillator 1 coated with the coupling agent 4. That is, the crystal oscillator 1 is immersed in an aqueous solution containing the nucleotide pair 6, and the phosphate group at the 5′-terminal of the nucleotide chain 5 and the OH of the coupling agent in the presence of carbodiimide.
The nucleotide chain 5 was fixed to the surface of the electrode 3 by binding with a group.
【００１８】また、この様なヌクレオチド鎖５の長さは
検出目的遺伝子を認識できる程度であればよい。そし
て、ヌクレオチド鎖５、５'からなるヌクレオチド対６
は、例えばＤＮＡ／ＲＮＡシンセサイザーを用いて合成
した後、耐熱性ポリメラーゼを用いたＰＣＲ装置によ
り、必要量（例えば１０万倍）まで増幅して使用する。The length of such a nucleotide chain 5 may be such that it can recognize the target gene for detection. And nucleotide pair 6 consisting of nucleotide chains 5 and 5 '.
Is synthesized, for example, using a DNA / RNA synthesizer, and then amplified by a PCR device using a thermostable polymerase up to the required amount (for example, 100,000 times) before use.
【００１９】上述のようにして電極３の表面にヌクレオ
チド鎖５との相補結合を介して固定化されたヌクレオチ
ド鎖５'にはポリスチレン等の修飾物質７が結合され
る。すなわち、一個あたり数個のメチロール基を導入し
たポリスチレン粒子を含む溶液をヌクレオチド対６が固
定された水晶発振子１に接触させた後、カルボジイミド
を加えてヌクレオチド鎖５'の５'−末端のリン酸基とポ
リスチレン粒子のメチロール基のＯＨとを結合させる。A modifying substance 7 such as polystyrene is bonded to the nucleotide chain 5'which is immobilized on the surface of the electrode 3 through the complementary bond with the nucleotide chain 5 as described above. That is, a solution containing polystyrene particles into which several methylol groups are introduced is brought into contact with the crystal oscillator 1 on which the nucleotide pair 6 is fixed, and then carbodiimide is added to add phosphorus at the 5′-end of the nucleotide chain 5 ′. The acid group is bonded to the OH of the methylol group of the polystyrene particles.
【００２０】以上のようにして得られた本発明の遺伝子
センサー１０は、検出目的遺伝子を次のようにして検出
する（第２図参照）。すなわち、検出目的ＤＮＡを含む
被検液に遺伝子センサー１０を接触させ、検出目的遺伝
子１１が遺伝子センサーに結合しやすいよう、相補的結
合を形成している水素結合を弱めるために加熱して、例
えば９０℃以上とする。このようにすると、ヌクレトチ
ド対６および検出目的遺伝子１１の相補的結合が解離す
る。その後、徐々に温度を下げてアニールすることで修
飾物質７で修飾されたヌクレトチド鎖５'と検出目的遺
伝子１１が置換して水晶発振子１の電極３表面に検出目
的遺伝子１１が結合する。The gene sensor 10 of the present invention obtained as described above detects a target gene for detection as follows (see FIG. 2). That is, the gene sensor 10 is brought into contact with a test solution containing DNA to be detected and heated to weaken the hydrogen bond forming a complementary bond so that the gene 11 to be detected can be easily bound to the gene sensor. 90 ° C or higher. By doing so, the complementary bond between the nucleotide pair 6 and the target gene 11 for detection is dissociated. Then, by gradually lowering the temperature and annealing, the nucleotide target 5 for detection modified with the modifying substance 7 is replaced with the target gene 11 for detection, and the target gene 11 for detection is bound to the surface of the electrode 3 of the crystal oscillator 1.
【００２１】すると、電極３の質量が減少し、水晶発振
子１の発振周波数が上昇する。この周波数の上昇によっ
て被検液中の検出目的遺伝子１１の存在が確認される。
同時に電極３表面への非特異的物質の吸着も起こるが、
これらは発振周波数を低下させる方向に働くので本発明
の遺伝子センサー１０は非常に選択性の高いものとな
る。Then, the mass of the electrode 3 is reduced and the oscillation frequency of the crystal oscillator 1 is increased. The presence of the detection target gene 11 in the test liquid is confirmed by this increase in frequency.
At the same time, adsorption of non-specific substances on the surface of the electrode 3 also occurs,
Since these act in the direction of lowering the oscillation frequency, the gene sensor 10 of the present invention has extremely high selectivity.
【００２２】水晶発振子１の周波数の上昇は、例えば第
３図に示す測定システムによって検知され、表示あるい
は出力される。すなわち、発振回路２０によって与えら
れた電圧に基づいて水晶発振子１０が発振する。発振し
た周波数信号は発振回路２０で増幅して周波数カウンタ
ー３０により周波数を算出しＡ／Ｄ変換した後、パソコ
ンに信号を送り、検出前の発振周波数と検出後の発振周
波数とを比較し、周波数の上昇データより検出目的遺伝
子の存在を判定する。判定された結果は、ディスプレイ
装置５０あるいは出力装置６０へ送られ表示あるいは印
字出力される。さらに、本発明の遺伝子センサーは、発
振周波数の変化が、検出目的遺伝子の濃度に対応して変
化するので、遺伝子の定量的な検出も可能である。The increase in the frequency of the crystal oscillator 1 is detected and displayed or output by the measuring system shown in FIG. 3, for example. That is, the crystal oscillator 10 oscillates based on the voltage given by the oscillation circuit 20. The oscillated frequency signal is amplified by the oscillating circuit 20, the frequency is calculated by the frequency counter 30, A / D converted, and then a signal is sent to the personal computer to compare the oscillating frequency before the detection with the oscillating frequency after the detection. The presence of the target gene for detection is determined from the elevation data of. The determined result is sent to the display device 50 or the output device 60 and displayed or printed out. Furthermore, in the gene sensor of the present invention, since the change in the oscillation frequency changes in accordance with the concentration of the detection target gene, the gene can be detected quantitatively.
【００２３】また、本発明の遺伝子センサー１０は、修
飾物質７として例えばフェライト等の磁性粒子を用いた
場合、修飾物質７結合被修飾ヌクレトチド鎖５'は使用
後、容易に回収することができ、遺伝子センサー１０の
材料として再生利用できる。In the gene sensor 10 of the present invention, when magnetic particles such as ferrite are used as the modifier 7, the modifier 7-bonded modified nucleotide chain 5'can be easily recovered after use, It can be recycled as a material for the gene sensor 10.
【００２４】[0024]
（実施例１）直径１２ｍｍ、厚さ０.３３４ｍｍの水晶
発振子（（株）明電舎製 ＡＴカット水晶円板）の両面
には、発振励起用の直径５ｍｍの金円板電極が形成され
ている。トルエンを溶媒としてシランカップリング剤４
（γ−グリシドキシプロピルシラン、ＳＨ６０４０、東
レ・ダウコーニング・シリコーン（株））の希釈液を作
成し、この希釈液をマイクロシリンジにとって金電極の
表面に１μｌ塗布した後、乾燥することによって被覆し
た。(Example 1) On both sides of a crystal oscillator having a diameter of 12 mm and a thickness of 0.334 mm (AT cut crystal disk manufactured by Meidensha Co., Ltd.), gold disk electrodes having a diameter of 5 mm for oscillation excitation are formed. . Silane coupling agent 4 using toluene as a solvent
(Γ-glycidoxypropylsilane, SH6040, Toray Dow Corning Silicone Co., Ltd.) is used to prepare a diluting solution, and 1 μl of the diluting solution is applied to the surface of the gold electrode by a microsyringe and then dried. did.
【００２５】この水晶発振子をＰＨ８.０の検出目的Ｄ
ＮＡの少なくとも一部分と同配列を持つヌクレオチド対
（本実施例においては試験例１で後述する方法によって
作成され用いられた検出目的ＤＮＡと同一のものをヌク
レオチド対として用いた）を含む溶液に浸漬し、カルボ
ジイミド存在下で、ヌクレオチド対の一方のヌクレオチ
ド鎖であるヌクレオチド鎖の５'−末端のリン酸基とシ
ランカップリング剤のＯＨ基とを結合して、水晶発振子
の電極表面に固定化した。This crystal oscillator is used for detection of PH 8.0 D
Immersion in a solution containing a nucleotide pair having the same sequence as at least a portion of NA (in this example, the same nucleotide as the detection target DNA prepared and used by the method described later in Test Example 1 was used) In the presence of carbodiimide, the phosphate group at the 5'-end of the nucleotide chain, which is one of the nucleotide chains of the nucleotide pair, and the OH group of the silane coupling agent were bound and immobilized on the electrode surface of the crystal oscillator. .
【００２６】次にヌクレオチド鎖と相補結合している反
対側のヌクレオチド鎖の５'−末端のリン酸基に、平均
質量が約１ｐｇであるポリスチレン微粒子（ポリサイエ
ンス社製 ポリビーズ（マイクロスフェア）平均粒径１
μｍ）に該ポリスチレン微粒子１個あたり数個のメチロ
ール基を導入したものを含む溶液を接触させ、カルボジ
イミドを加えて該ポリスチレン微粒子に導入されたメチ
ロール基のＯＨ基を２４時間反応させることによって結
合した。これを洗浄することによって、ＤＮＡセンサー
とした。Next, polystyrene fine particles having an average mass of about 1 pg in the phosphate group at the 5'-end of the opposite nucleotide chain complementary to the nucleotide chain (polybeads (microspheres) average particle manufactured by Polyscience Co., Ltd.) Diameter 1
μm) was brought into contact with a solution containing a plurality of methylol groups introduced per polystyrene fine particle, carbodiimide was added, and the OH groups of the methylol groups introduced into the polystyrene fine particles were reacted for 24 hours for binding. . This was washed to obtain a DNA sensor.
【００２７】（試験例１）このようにして得られたＤＮ
Ａセンサーを用いて実際に被検液中のＤＮＡの存在を確
認する実験を行った。試料ＤＮＡは、ＤＮＡ／ＲＮＡ
SYNTHESIZERS 392型（Applied Biosystems Inc.社製）
のＤＮＡシンセサイザーを用いて合成した後、耐熱性Ｄ
ＮＡポリメラーゼを用いたＰＣＲ装置（ＣＩＴＵＳ社
製）により１０万倍に増幅した後使用した。(Test Example 1) DN thus obtained
An experiment was carried out to actually confirm the presence of DNA in the test solution using the A sensor. Sample DNA is DNA / RNA
SYNTHESIZERS 392 type (manufactured by Applied Biosystems Inc.)
Heat resistance D after synthesis using the DNA synthesizer
It was used after being amplified 100,000 times by a PCR device (manufactured by CITUS) using NA polymerase.
【００２８】実施例１で得られたＤＮＡセンサーを第３
図に示す測定システムに接続した。尚、周波数カウンタ
ー３０は横河ヒューレットパッカード社製ＹＨＰ−５３
３４を使用した。乾燥空気中で発振周波数を測定した結
果、5,001,906Ｈｚであった。この水晶発振子の基本発
振周波数は5,002,201Ｈｚであり、基本発振周波数より
２９５Ｈｚ低下している。The DNA sensor obtained in Example 1 was used as the third sensor.
It was connected to the measurement system shown in the figure. The frequency counter 30 is YHP-53 manufactured by Yokogawa Hewlett-Packard Company.
34 was used. As a result of measuring the oscillation frequency in dry air, it was 5,001,906 Hz. The fundamental oscillation frequency of this crystal oscillator is 5,002,201 Hz, which is 295 Hz lower than the fundamental oscillation frequency.
【００２９】また、このＤＮＡセンサーを試料ＤＮＡを
１×１０5分子含む被検液１００μｌに接触させて、溶
液温度を９３℃で１分間保持した後、ゆっくりと降温し
ながらアニールを行った。その後、被検液からＤＮＡセ
ンサーを取り出して洗浄し、乾燥空気中で発振周波数を
測定すると5,001,936Ｈｚであり、３０Ｈｚの周波数上
昇が観察された。The DNA sensor was brought into contact with 100 μl of a test solution containing 1 × 10 5 molecules of sample DNA, the solution temperature was maintained at 93 ° C. for 1 minute, and then annealing was performed while slowly lowering the temperature. Thereafter, the DNA sensor was taken out from the test liquid, washed, and the oscillation frequency was measured in dry air to be 5,001,936 Hz, and a frequency increase of 30 Hz was observed.
【００３０】ところで、基本発振周波数Ｆ0は水晶板の
厚さｄで決まり、厚くなれば周波数は低下し、この関係
は次式で与えられる。Ｆ0＝Ｎ／ｄ （Ｎ：振動定数、
本明細書中で使用したものは167kHz・cm）。このことは
水晶の表面に一定質量のものが被着して厚みが変わって
も同様に周波数が低下することを意味し、被着物質の質
量ｍと発振周波数のシフト値ΔＦとの関係は次式で与え
られる。ｍ＝-4.348・10-7・A・ΔF/F0 2 （Ａ：電極面
積）。実施例１のＤＮＡセンサーでは、基本発振周波数
Ｆ0＝（167/334）＝５×１０3ｋＨｚ＝５ＭＨｚ、電極
面積Ａ＝(0.5/2)2×3.14＝０.１９６ｃｍ2となる。By the way, the fundamental oscillation frequency F 0 is determined by the thickness d of the crystal plate, and as the thickness becomes thicker, the frequency decreases. This relationship is given by the following equation. F 0 = N / d (N: vibration constant,
The one used in this specification is 167 kHz · cm). This means that even if the material of constant mass is deposited on the surface of the crystal and the thickness is changed, the frequency is similarly reduced, and the relationship between the mass m of the deposited material and the shift value ΔF of the oscillation frequency is as follows. Given by the formula. m = -4.348 · 10 −7 · A · ΔF / F 0 2 (A: electrode area). In the DNA sensor of Example 1, the fundamental oscillation frequency F 0 = (167/334) = 5 × 10 3 kHz = 5 MHz, and the electrode area A = (0.5 / 2) 2 × 3.14 = 0.196 cm 2 .
【００３１】また、ＤＮＡセンサーに固定されたポリス
チレン微粒子結合ヌクレオチド鎖は、ｍ＝１×１０-6ｇ
であり、上述の通りポリスチレン微粒子１個の質量は１
ｐｇであるから、１×１０6個であることがわかった。Further, the polystyrene fine particle-bonded nucleotide chain immobilized on the DNA sensor has m = 1 × 10 −6 g
As described above, the mass of one polystyrene fine particle is 1
Since it is pg, it was found to be 1 × 10 6 .
【００３２】被検液中の試料ＤＮＡの測定によって得ら
れた周波数変化ΔＦ＝３０Hzからは、ｍ＝−１.０２×
１０-7ｇであることがわかり、−１.０２×１０5個のポ
リスチレン微粒子結合ヌクレオチド鎖が外れたことがわ
かる。また、１００μｌ中の試料ＤＮＡの分子数を２×
１０5、５×１０4個としたときのΔＦはそれぞれ６１、
１４Ｈｚであり、ほぼ定量的な特性を示すことがわかっ
た。From the frequency change ΔF = 30 Hz obtained by measuring the sample DNA in the test solution, m = -1.02 ×
It was found to be 10 −7 g, and it was found that −1.02 × 10 5 polystyrene fine particle-bonded nucleotide chains were detached. In addition, the number of molecules of the sample DNA in 100 μl is 2 ×
ΔF when it is 10 5 , 5 × 10 4 is 61,
It was 14 Hz, and it was found that the characteristics were almost quantitative.
【００３３】また、実施例１のＤＮＡセンサーの検出限
界は、通常の周波数測定では１Ｈｚの測定精度は容易に
得られるので、発振周波数を１Ｈｚだけ変化させられる
質量をこのセンサーの質量感度とすると、この場合は
３.４ｎｇ／Ｈｚとなり、これより１分子あたり１ｐｇ
の変化が起きるから、３４００分子であることがわかっ
た。The detection limit of the DNA sensor of Example 1 is that a measurement accuracy of 1 Hz can be easily obtained in normal frequency measurement. Therefore, when the mass whose oscillation frequency can be changed by 1 Hz is the mass sensitivity of this sensor, In this case, it will be 3.4 ng / Hz, which is 1 pg per molecule.
Was found to be 3400 molecules.
【００３４】（実施例２）基本発振周波数が９.９５Ｍ
Ｈｚである直径３ｍｍ金電極をもつ直径８ｍｍの水晶発
振子を用いた以外は実施例１と同様にしてＤＮＡセンサ
ーを作製した。本実施例では約３０６個からのＤＮＡ検
出が可能である。(Embodiment 2) The fundamental oscillation frequency is 9.95M.
A DNA sensor was produced in the same manner as in Example 1 except that a crystal oscillator having a diameter of 8 mm and a gold electrode having a diameter of 3 mm was used. In this example, about 306 DNAs can be detected.
【００３５】（実施例３）基本発振周波数２０ＭＨｚで
ある直径２ｍｍの金電極をもつ直径４ｍｍの水晶発振子
を用いた以外は実施例１と同様にしてＤＮＡセンサーを
作製した。本実施例では３４から個のＤＮＡ検出が可能
である。Example 3 A DNA sensor was prepared in the same manner as in Example 1 except that a crystal oscillator having a diameter of 4 mm and a gold electrode having a diameter of 2 mm and a fundamental oscillation frequency of 20 MHz was used. In this embodiment, 34 DNAs can be detected.
【００３６】（実施例４）基本発振周波数５０ＭＨｚで
ある直径１ｍｍの金電極をもつ直径２ｍｍの水晶発振子
を用いた以外は実施例１と同様にしてＤＮＡセンサーを
作製した。本実施例では１個からのＤＮＡ検出が可能で
ある。Example 4 A DNA sensor was prepared in the same manner as in Example 1 except that a crystal oscillator having a diameter of 2 mm and a gold electrode having a diameter of 1 mm having a fundamental oscillation frequency of 50 MHz was used. In this example, it is possible to detect DNA from one DNA.
【００３７】[0037]
【発明の効果】以上の説明から明らかなように、本発明
の遺伝子センサーは、検出目的遺伝子よりも大きな質量
をもつ物質があらかじめ固定され、検出目的遺伝子がこ
れと置換してセンサーに結合する質量減少方式であるの
で、高感度で、非特異的物質の吸着からの選択性が高い
遺伝子センサーとなる。また本発明は、放射線標識や酵
素等を用いていないので取り扱いが簡単かつ、安全であ
る。また、操作が簡単である。従来法に比較して迅速測
定が可能である。本発明によれば、目的の遺伝子の一部
の配列が分かれば高感度で遺伝子を検出することが可能
であり、ＨＩＶウイルス、ＨＣＶウイルス等を遺伝子レ
ベルで検出することができ遺伝子レベルの病因の診断や
治療に貢献できる。As is apparent from the above description, in the gene sensor of the present invention, a substance having a mass larger than that of the target gene of detection is immobilized in advance, and the target gene of interest replaces this substance to bind to the sensor. Since the reduction method is used, the gene sensor has high sensitivity and high selectivity from adsorption of non-specific substances. Further, since the present invention does not use a radiolabel or an enzyme, it is easy to handle and safe. Moreover, the operation is simple. Rapid measurement is possible compared with the conventional method. According to the present invention, it is possible to detect a gene with high sensitivity if a part of the sequence of the target gene is known, and it is possible to detect HIV virus, HCV virus, etc. at the gene level and Can contribute to diagnosis and treatment.
【００３８】また、本発明の検出部分は、再生可能なの
で、繰り返し使用して検査コストを低くできる。本発明
の遺伝子センサーを複数個配列して形成することで同時
に多種類検査ができる。Since the detecting portion of the present invention is reproducible, the inspection cost can be reduced by repeatedly using it. By arranging and forming a plurality of gene sensors of the present invention, multiple types of tests can be performed simultaneously.
第１図は、本発明の１実施例による遺伝子センサーの断
面構造を示す模式図である。第２図は、本発明の遺伝子
検出原理を説明する図である。第３図は、本発明の遺伝
子センサーを組み込んだ遺伝子検出システムの構成図で
ある。FIG. 1 is a schematic diagram showing a cross-sectional structure of a gene sensor according to one example of the present invention. FIG. 2 is a diagram explaining the principle of gene detection of the present invention. FIG. 3 is a block diagram of a gene detection system incorporating the gene sensor of the present invention.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｑ 1/68 Ａ 7823−4Ｂ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI technical display location C12Q 1/68 A 7823-4B
Claims (1)
出目的遺伝子と相補的な塩基配列を有する該電気機械変
換素子の電極表面に固定された第１のヌクレオチド鎖
と、該第１のヌクレオチド鎖と相補結合している第２の
ヌクレオチド鎖とを有する遺伝子センサーであって、該
第２のヌクレオチド鎖には検出目的遺伝子よりも大きな
質量をもつ物質が固定されてなることを特徴とする遺伝
子センサ−。1. An electromechanical conversion element, a first nucleotide chain at least a part of which has a base sequence complementary to a detection target gene and which is fixed to the electrode surface of the electromechanical conversion element, and the first nucleotide. A gene sensor having a second nucleotide chain complementary to a chain, wherein a substance having a mass larger than that of a target gene for detection is immobilized on the second nucleotide chain. Sensor.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23860792A JP3296854B2 (en) | 1992-09-07 | 1992-09-07 | Gene sensor |
| EP93307056A EP0587408A1 (en) | 1992-09-07 | 1993-09-07 | Method for determination of DNA and sensor therefor |
| US08/117,329 US5552274A (en) | 1992-09-07 | 1993-09-07 | Method for detecting target sequences by oscillation frequency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23860792A JP3296854B2 (en) | 1992-09-07 | 1992-09-07 | Gene sensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0688779A true JPH0688779A (en) | 1994-03-29 |
| JP3296854B2 JP3296854B2 (en) | 2002-07-02 |
Family
ID=17032695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23860792A Expired - Fee Related JP3296854B2 (en) | 1992-09-07 | 1992-09-07 | Gene sensor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3296854B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006517668A (en) * | 2003-02-12 | 2006-07-27 | アッタナ アーベー | Piezoelectric resonator |
| WO2007018187A1 (en) * | 2005-08-05 | 2007-02-15 | Kyowa Medex Co., Ltd. | Measurement instrument, measurement kit using the same, measurement method, measurement device, and piezoelectric oscillator reproducing method |
| WO2011111785A1 (en) * | 2010-03-10 | 2011-09-15 | 国立大学法人東京工業大学 | Laminated structure for measurement of intensity of reflected light, device equipped with laminated structure for measurement of reflected light, and method for determination of thickness and/or mass and/or viscosity of thin film |
-
1992
- 1992-09-07 JP JP23860792A patent/JP3296854B2/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006517668A (en) * | 2003-02-12 | 2006-07-27 | アッタナ アーベー | Piezoelectric resonator |
| JP2010237218A (en) * | 2003-02-12 | 2010-10-21 | Attana Ab | Piezoelectric resonator |
| US9762204B2 (en) | 2003-02-12 | 2017-09-12 | Attana Ab | Piezoelectric resonator |
| WO2007018187A1 (en) * | 2005-08-05 | 2007-02-15 | Kyowa Medex Co., Ltd. | Measurement instrument, measurement kit using the same, measurement method, measurement device, and piezoelectric oscillator reproducing method |
| WO2011111785A1 (en) * | 2010-03-10 | 2011-09-15 | 国立大学法人東京工業大学 | Laminated structure for measurement of intensity of reflected light, device equipped with laminated structure for measurement of reflected light, and method for determination of thickness and/or mass and/or viscosity of thin film |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3296854B2 (en) | 2002-07-02 |
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