JP2006234686A - Chip for detecting small mass - Google Patents

Chip for detecting small mass Download PDF

Info

Publication number
JP2006234686A
JP2006234686A JP2005051798A JP2005051798A JP2006234686A JP 2006234686 A JP2006234686 A JP 2006234686A JP 2005051798 A JP2005051798 A JP 2005051798A JP 2005051798 A JP2005051798 A JP 2005051798A JP 2006234686 A JP2006234686 A JP 2006234686A
Authority
JP
Japan
Prior art keywords
mass detection
detection chip
reaction
electrode
micro
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
Application number
JP2005051798A
Other languages
Japanese (ja)
Other versions
JP4597710B2 (en
Inventor
Motoyasu Hanji
元康 判治
Masako Takada
雅子 高田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kyocera Crystal Device Corp
Original Assignee
Kyocera Crystal Device Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Kyocera Crystal Device Corp filed Critical Kyocera Crystal Device Corp
Priority to JP2005051798A priority Critical patent/JP4597710B2/en
Publication of JP2006234686A publication Critical patent/JP2006234686A/en
Application granted granted Critical
Publication of JP4597710B2 publication Critical patent/JP4597710B2/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip for detecting small mass related to the extraction electrode of an integrated chip for detecting the small mass for detecting the state of the reaction, which occurs on an exciting electrode by forming a ligand on the exciting electrode, as a change in mass and to easily inspect DNA. <P>SOLUTION: In the chip constituted by forming the exciting electrodes to the upper and under surfaces of the recesses of a piezoelectric single crystal substrate equipped with a plurality of the recesses respectively separated at predetermined intervals and setting the electrodes on one sides of the recesses as reaction electrodes to form the ligand, which captures a reaction state, on the reaction electrodes, a grooved flow channel is formed to a part of the piezoelectric single crystal substrate forming the recesses and a reagent is injected from the flow channel. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、励振電極上にリガンドを形成し、その上で発生する反応の状態を、質量の変化としてリアルタイムに検出する集積型微少質量検出チップの薬液を注入する流路形状に関するものである。   The present invention relates to a flow channel shape for injecting a chemical solution of an integrated micro mass detection chip that forms a ligand on an excitation electrode and detects a reaction state generated on the ligand in real time as a change in mass.

本明細書の使用方法を説明するにあたり、ＤＮＡチップを一例として説明する。近年、人の遺伝子構造がほぼ解明され、テーラーメイド医療などを目的に解明された遺伝子の持つ機能の調査，研究が本格化してきている。この遺伝子の機能解明の一手法として、ＤＮＡチップが用いられている。ＤＮＡは、Ａ（アデニン），Ｔ（チミン），Ｃ（シトシン），Ｇ（グアニン）の４つの塩基により形成された２本の分子鎖が、螺旋状に結合して形成されている。４つの塩基は、それぞれ結合することが可能で、その組み合わせはＡとＴ、ＣとＧとなっている。   In describing the usage method of the present specification, a DNA chip will be described as an example. In recent years, the genetic structure of humans has been almost elucidated, and the investigation and research of the functions of genes elucidated for the purpose of tailor-made medicine has been in full swing. As a technique for elucidating the function of this gene, a DNA chip is used. DNA is formed by combining two molecular chains formed by four bases of A (adenine), T (thymine), C (cytosine), and G (guanine) in a spiral shape. Four bases can be bonded to each other, and the combinations are A and T, and C and G.

従来、ＤＮＡチップは、平板のガラスやシリコンなどの基板上に、高密度にＤＮＡ分子の断片を固定したものである。そして、検体となるＤＮＡを被検者の血液から抽出液中に抽出し、その溶液中のＤＮＡを１本鎖に分離切断した後、抽出溶液を含めてＤＮＡチップの表面に滴下すると、ＤＮＡチップ上に構成されたＤＮＡ配列に対応して検体ＤＮＡが結合するので、その結合状態を蛍光変化として判定している。   Conventionally, a DNA chip is obtained by fixing DNA molecule fragments at high density on a substrate such as flat glass or silicon. Then, the sample DNA is extracted from the blood of the subject into the extract, the DNA in the solution is separated and cut into single strands, and then dropped onto the surface of the DNA chip including the extract solution. Since the sample DNA binds corresponding to the DNA sequence configured above, the binding state is determined as the fluorescence change.

則ち、現在実用化されているＤＮＡチップを用いた検出方式で、レーザを照射して蛍光を測定する蛍光検出方式である。この方式では、検体となるＤＮＡに予め蛍光色素で標識をつけ、ＤＮＡチップ上のＤＮＡ断片に結合した検体ＤＮＡの有無を、レーザ光照射による蛍光色素の発光により検出する方法で、判定までには多くの時間を必要とし、医療現場など緊急判断には問題である。（非特許文献１参照）。   In other words, this is a fluorescence detection method that uses a DNA chip that is currently in practical use and measures fluorescence by irradiating a laser. In this method, the sample DNA is labeled with a fluorescent dye in advance, and the presence or absence of the sample DNA bound to the DNA fragment on the DNA chip is detected by the emission of the fluorescent dye by laser light irradiation. It takes a lot of time and is a problem for emergency judgments such as in the medical field. (Refer nonpatent literature 1).

これに対して本願出願人は、リアルタイム計測を目的として既に出願している特許文献１に示す様な水晶基板を用いたＤＮＡチップを提案している。
このＤＮＡチップは水晶基板に形成された各々分離した複数の例えば凸部、凹部ら成る島部（セル）と、その上に構成された電極膜に構築された特有の塩基配列を持つＤＮＡ群から構成され、このＤＮＡチップを溶液中で交流電圧を印加して励振させ、その励振周波数を確認しながら、検体から検出されたＤＮＡを含む溶液を注入するとＤＮＡ相互間の結合の状態により、電極上の質量が微小変化するため、励振周波数が変化する。また、この結合の様子は周波数を繰り返し計測することでリアルタイムに観測することが可能となる。 On the other hand, the applicant of the present application has proposed a DNA chip using a quartz substrate as shown in Patent Document 1 already applied for the purpose of real-time measurement.
This DNA chip is composed of a plurality of separated islands (cells) formed on a quartz substrate, for example, convex portions and concave portions, and a DNA group having a unique base sequence constructed on an electrode film formed thereon. This DNA chip is excited by applying an alternating voltage in the solution, and when a solution containing DNA detected from the specimen is injected while checking the excitation frequency, Since the mass of the material changes minutely, the excitation frequency changes. In addition, the state of this coupling can be observed in real time by repeatedly measuring the frequency.

このとき、各セルの励振電極はそれぞれ独立した引き出し電極により端子に接続とれており、各セルが独立で励振できるような配線構造をとっている。以上のように、圧電式微小質量計測センサは、例えば水晶振動子表面に電極を形成し、この膜表面上で物質を脱着することにより質量変化を周波数変化として捉える手法であり、この現象をサブレーの式から算出している。   At this time, the excitation electrode of each cell is connected to the terminal by an independent lead electrode, and a wiring structure is adopted so that each cell can be excited independently. As described above, the piezoelectric micromass measuring sensor is a method of capturing a mass change as a frequency change by, for example, forming an electrode on the surface of a crystal resonator and desorbing a substance on the film surface. It is calculated from the formula.

原田 学，佐藤 高遠，米田 英克、「ＤＮＡチップの現状と展望」、応用物理、第６９巻、第１２号（２０００）Manabu Harada, Takato Sato, Hidekatsu Yoneda, “Current Status and Prospects of DNA Chips”, Applied Physics, Vol. 69, No. 12 (2000) 特開２００３−２８７５３８号公報 なお出願人は前記した先行技術文献情報で特定される先行技術文献以外には、本発明に関連する先行技術文献を、本件出願時までに発見するに至らなかった。JP, 2003-287538, A The applicant did not come to discover prior art documents relevant to the present invention by the time of this application other than the prior art documents specified by the prior art document information described above.

しかしながら、前述したような、従来の技術では、レーザ照射装置など大がかりな装置が必要となり、システムが高価なものとなる。また、この様な手法では、反応状態を定量的に検出することが容易ではない。本発明は、以上のような問題点を解消するためになされたものであり、定量的測定、リアルタイムに容易に異なる複数種のＤＮＡの検出ができるようにすることを目的とし、更なるＤＮＡチップの利便性を高めたＤＮＡチップ構造となっている。   However, in the conventional technology as described above, a large-scale device such as a laser irradiation device is required, and the system becomes expensive. In addition, with such a method, it is not easy to quantitatively detect the reaction state. The present invention has been made to solve the above-described problems, and aims to enable quantitative measurement and easy detection of a plurality of different types of DNA in real time. It has a DNA chip structure with improved convenience.

その具体的な特徴としては、圧電素子を使用して、ＤＮＡの合致性、抗原、抗体反応などを質量変化で観測する場合センサ部の片側を溶液中に露出する必要がある。このとき、溶液に接する電極（反応側電極）は、センサ素子の外周部を経由して気相側に引き出した後、気相環境においてリード線を介して発振器や計測機器などに接続される。しかし例えばマトリックス状のセルで構成した集積型反応解析では、それぞれのセルを逆メサ構造で構築することが最も有効な構造と考える。   As a specific feature thereof, when using a piezoelectric element to observe DNA consistency, antigen, antibody reaction, etc. by mass change, it is necessary to expose one side of the sensor part in the solution. At this time, an electrode in contact with the solution (reaction side electrode) is pulled out to the gas phase side through the outer peripheral portion of the sensor element, and then connected to an oscillator, a measuring instrument, or the like via a lead wire in the gas phase environment. However, for example, in integrated reaction analysis composed of matrix cells, it is considered that the most effective structure is to construct each cell with an inverted mesa structure.

上述する微少質量検出チップを用いた場合、計測時の試薬、媒質溶液が少量である場合、試薬の流れにムラがあったり、また凹部を備えた圧電単結晶基板の微少質量検出チップの場合にあっては、溶液を入れ替える過程において、凹部の角部に試薬が残存することも考えられ、試薬溶液中の反応が均一に行き渡らないなどの課題がある。   When the above-mentioned micro mass detection chip is used, if the reagent and medium solution at the time of measurement are small, the flow of the reagent is uneven, or the micro mass detection chip of the piezoelectric single crystal substrate having a recess is used. In this case, in the process of replacing the solution, the reagent may remain at the corners of the recess, and there is a problem that the reaction in the reagent solution does not spread uniformly.

上述する課題を解決するために本発明は、所定の間隔でそれぞれ分離した複数の凹部を備えた圧電単結晶基板の凹部の表裏に励振電極を形成し、一方面の電極を反応電極としそれぞれの反応側電極上に反応状態を捉えるリガンドを形成した微少質量検出チップにおいて、前記凹部を形成する圧電単結晶基板の一部に溝部の流路を形成し、前記流路から試薬を注入することを特徴とする微少質量検出チップである。   In order to solve the above-described problems, the present invention forms excitation electrodes on the front and back sides of a concave portion of a piezoelectric single crystal substrate having a plurality of concave portions that are separated at predetermined intervals, and each side electrode serves as a reaction electrode. In a minute mass detection chip in which a ligand that captures a reaction state is formed on a reaction side electrode, a groove channel is formed in a part of the piezoelectric single crystal substrate that forms the recess, and a reagent is injected from the channel. This is a feature of a minute mass detection chip.

そして、その微少質量検出チップにおいては、個々の微少質量検出チップの前記圧電単結晶基板に形成する溝部の流路の凹部内側の一方には、２稜線に略１２０°拡がる流路があり、他方の流路にも２稜線に略１２０°拡がる流路により他方の前記流路に連結した微少質量検出チップが連続した形態を有している。   And in the minute mass detection chip, one of the inner sides of the groove of the groove part formed in the piezoelectric single crystal substrate of each of the minute mass detection chips has a channel extending approximately 120 ° on two ridge lines, This flow path also has a form in which a minute mass detection chip connected to the other flow path is continuous by a flow path extending about 120 ° along two ridge lines.

従って、所定の間隔で各々分離した複数の凹部を備えた水晶基板と、前記凹部に相当部分の表裏には電極を形成し、特有な塩基配列を持つ複数の１本鎖ＤＮＡから構成されており、前記ＤＮＡチップの励振電極を形成する一方の引き出し電極を、前記ＤＮＡチップの主面の一部にスルーホールを形成して対向面側に引き出し電極をＤＮＡチップの一方側に導出した構造であり、実際の測定時にはスルーホールをシール材で埋めた形状となっている。   Therefore, it is composed of a quartz substrate having a plurality of recesses separated at predetermined intervals, and a plurality of single-stranded DNAs having unique base sequences, with electrodes formed on the front and back of the corresponding portions of the recesses. The structure is such that one extraction electrode forming the excitation electrode of the DNA chip has a through hole formed in a part of the main surface of the DNA chip and the extraction electrode is led out to one side of the DNA chip on the opposite surface side. In actual measurement, the through hole is filled with a sealing material.

また、ＤＮＡチップとしては前記凹部上には金薄膜電極が形成され、その表面を親水性にする親水処理を施した領域を構築し、更に前記ＤＮＡ断片は、一端がＳＨ基に置換され、前記ＳＨ基を介して前記ＤＮＡ断片が前記金薄膜に固定されていることを特徴とするＤＮＡチップである。   In addition, as a DNA chip, a gold thin film electrode is formed on the recess, and a region subjected to a hydrophilic treatment for making the surface hydrophilic is constructed. Further, one end of the DNA fragment is replaced with an SH group, The DNA chip is characterized in that the DNA fragment is fixed to the gold thin film via an SH group.

上述する本発明のＤＮＡ検査方法の原理としては、所定の間隔で各々分離した複数の凸部や凹部などからなるセルを備えた水晶基板を用意し、例えば各々の凹部上に標識された特有の塩基配列から構成されたリガンド（ＤＮＡ）断片を固定し、溶液中でＤＮＡ断片が固定された状態の各々凹部の共振周波数を測定して凹部各々の第１の測定周波数とし、次ぎに検体となるＤＮＡ（狙いとするＤＮＡ）を含んだ溶液中に所定時間接触させ、各々の凹部の共振周波数を測定して凹部各々の第２の測定周波数とし、第２の測定周波数と第１の測定周波数との差が発生することにより、検体となるＤＮＡの中から標識されたＤＮＡ断片と同じ塩基配列のものを検出するようにしたものである。このＤＮＡ検査方法によれば、各凹部における第１の測定周波数と第２の測定周波数との差により、各凹部上に固定されているＤＮＡ断片に結合したＤＮＡの重量を検出することで実現する。   The principle of the above-described DNA testing method of the present invention is to prepare a quartz substrate provided with a cell composed of a plurality of convex portions and concave portions separated at a predetermined interval, for example, a unique label labeled on each concave portion. A ligand (DNA) fragment composed of a base sequence is immobilized, and the resonance frequency of each recess is measured in a state in which the DNA fragment is immobilized in a solution to obtain the first measurement frequency of each recess, and then a sample is obtained. Contact with a solution containing DNA (target DNA) for a predetermined period of time, measure the resonance frequency of each recess to be the second measurement frequency of each recess, the second measurement frequency and the first measurement frequency As a result, the same base sequence as that of the labeled DNA fragment is detected from the sample DNA. This DNA testing method is realized by detecting the weight of DNA bound to the DNA fragment fixed on each recess by the difference between the first measurement frequency and the second measurement frequency in each recess. .

また、反応進行中に繰り返し周波数を計測することにより、リアルタイムに反応状態を観測することが可能となるほか微少質量検出チップ同士を連続して接続した流路を形成することで、異なる特有の塩基配列のＤＮＡを用意しておき、同一検体ＤＮＡ溶液を流路を介して流すことで、複数の反応結果を得ることができる。   In addition, it is possible to observe the reaction state in real time by measuring the frequency repeatedly while the reaction is in progress. In addition, by forming a flow path in which minute mass detection chips are continuously connected, different specific bases are formed. A plurality of reaction results can be obtained by preparing DNA of sequence and flowing the same sample DNA solution through the flow path.

以上説明したように、本発明によれば、試薬溶液の乱流を防止することが出来、溶液残が発生しないので、確実に反応に対して溶液を分離して扱うことができるので計測信頼性が向上する。その結果サブレーの理論に基づいて測定結果を忠実に再現できることにより、公式をそのまま利用して精度良く計測が可能となる。また、凹部の液導入出口壁面を１２０°に開く形態を有する微少質量検出チップは、凹部角部に残留する試薬を無くすことにより、使用する試薬の変換量が少量でも液の置換が確実になり、精度の良い計測が可能となるため測定効率を向上することができる。   As described above, according to the present invention, the turbulent flow of the reagent solution can be prevented, and no solution residue is generated, so that the solution can be reliably separated and handled for the reaction. Will improve. As a result, the measurement result can be faithfully reproduced based on the sub-lay theory, so that the measurement can be performed with high accuracy using the formula as it is. In addition, a minute mass detection chip having a configuration in which the liquid inlet / outlet wall surface of the concave portion is opened at 120 ° eliminates the reagent remaining in the corner portion of the concave portion, thereby ensuring liquid replacement even with a small amount of reagent conversion. Measurement accuracy can be improved because accurate measurement is possible.

以下、本発明の実施の形態について図を参照して説明する。図１は、本発明の実施の形態における微小質量検出チップ（ＤＮＡチップ）の構成を示す斜視図（ａ）と断面図（ｂ）である。このＤＮＡチップは、一辺が２０〜２５ｍｍ程度、板厚０．１ｍｍ程度のＡＴカットの水晶基板１上に、直径１ｍｍ程度，深さ５０〜６０μｍ程度の複数の凹部２が、２ｍｍ間隔でマトリクス状に形成され、これら複数の凹部２上に、励振電極として金を表面を表面とするが、チタン−金合金の薄膜を形成し、励振時の周波数別に２５〜４０ＭＨｚ程度になるように凹部を加工し、形成された金薄膜３の表面に、リガンド（ＤＮＡ断片）が各々固定されているものである。   Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view (a) and a sectional view (b) showing a configuration of a minute mass detection chip (DNA chip) in an embodiment of the present invention. In this DNA chip, a plurality of recesses 2 having a diameter of about 1 mm and a depth of about 50 to 60 μm are formed in a matrix at intervals of 2 mm on an AT-cut quartz substrate 1 having a side of about 20 to 25 mm and a thickness of about 0.1 mm. The gold is used as the excitation electrode on the surface of the plurality of recesses 2, but a titanium-gold alloy thin film is formed, and the recesses are processed so that the frequency is about 25 to 40 MHz depending on the frequency during excitation. The ligand (DNA fragment) is fixed on the surface of the gold thin film 3 formed.

凹部２の上へのＤＮＡ断片の固定は、つぎに示すようにする。まず、所望とする１本鎖ＤＮＡ断片の一端をＳＨ基で置換された状態とする。次いで、ＳＨ基で一端が置換されたＤＮＡ断片が分散している溶媒中に、金薄膜３が各凹部２の表面に形成された水晶基板１を浸漬する。このことにより、金薄膜３の上にＳＨ基が引き寄せられて固着する。この結果、金薄膜３の表面にＳＨ基を介してＤＮＡ断片が固定された状態となる。この後、水晶基板１上の溶液はＰＢＳ（リン酸緩衝生理食塩水）で置換しておく。   The DNA fragment is fixed on the recess 2 as follows. First, one end of a desired single-stranded DNA fragment is in a state substituted with an SH group. Next, the quartz substrate 1 on which the gold thin film 3 is formed on the surface of each recess 2 is immersed in a solvent in which a DNA fragment whose one end is substituted with an SH group is dispersed. As a result, the SH group is attracted and fixed onto the gold thin film 3. As a result, the DNA fragment is fixed to the surface of the gold thin film 3 via the SH group. Thereafter, the solution on the quartz substrate 1 is replaced with PBS (phosphate buffered saline).

上述するＤＮＡチップの本願発明の特徴には、所定の間隔でそれぞれ分離した複数の凹部を備えた圧電単結晶基板の凹部の表裏に励振電極を形成し、一方面の電極を反応電極としそれぞれの反応側電極上に反応状態を捉えるリガンドを形成し、形成された励振電極の引き出し電極の一方を、前記チップの主面にスルーホ−ル４を形成しスルーホールを介して対面側に引き出したことを特徴とする微少質量検出チップである。また、複数個をひとつの圧電基板上に複数の励振電極（反応側電極）上に、それぞれ異なる塩基配列のＤＮＡ、或いは、ＲＮＡ断片（リガンド）を固定することで、各種の成分を同時に測定することもできる。   A feature of the present invention of the DNA chip described above is that excitation electrodes are formed on the front and back of the recesses of the piezoelectric single crystal substrate having a plurality of recesses separated at predetermined intervals, and the electrodes on one side are used as reaction electrodes. A ligand that captures the reaction state was formed on the reaction side electrode, and one of the lead electrodes of the formed excitation electrode was formed on the main surface of the chip through the through hole 4 and pulled out to the opposite side through the through hole. This is a minute mass detection chip characterized by the following. In addition, various components can be measured simultaneously by immobilizing a plurality of DNAs or RNA fragments (ligands) of different base sequences on a plurality of excitation electrodes (reaction side electrodes) on a single piezoelectric substrate. You can also.

要するに、図１の断面図に示すように、圧電単結晶基板の凹部の表裏に励振電極を形成し、凹部に形成する励振電極を圧電結晶基板の一部にスルーホール４を形成し、裏面へと引き出すことを特徴するものである。なお、図１では個々の素子を表現するために、圧電結晶基板の全体を二点鎖線で示している。   In short, as shown in the cross-sectional view of FIG. 1, excitation electrodes are formed on the front and back sides of the recesses of the piezoelectric single crystal substrate, and through holes 4 are formed in a part of the piezoelectric crystal substrate. It is characterized by drawing out. In FIG. 1, the entire piezoelectric crystal substrate is indicated by a two-dot chain line in order to express individual elements.

ここで、図１については、リガンドを形成する箇所が圧電結晶基板の凹部の底部に電極を形成したものであり、図２については、同様の構成ながら電極形成部を更に削りリガンドが底部の表面と同一面（高さ）になるようにしたものである。
このチップで使用する電極膜厚は数百ｎｍ程度のものが使用され、計測しようとするＤＮＡ分子数層分の厚みとなる。ここで重量−周波数変換のためのＳａｕｅｂｒｅｙの式（１式）をみると、 Here, in FIG. 1, the portion where the ligand is formed is an electrode formed on the bottom of the concave portion of the piezoelectric crystal substrate. In FIG. 2, the electrode forming portion is further scraped with the same configuration, but the ligand is on the bottom surface. And the same surface (height).
The electrode used in this chip has a thickness of about several hundreds of nanometers, which is the thickness of several layers of DNA molecules to be measured. Here, Sauebrey's equation (1) for weight-frequency conversion is

ｄＦ＝−（ｄＭＦ２）／（Ａρｋｆ） ・・・（１式）
ｄＦ２ ：周波数変化
ｄＭ ：質量変化
Ｆ ：励振周波数
Ａ ：電極面積
ρ ：水晶の密度
ｋｆ ：水晶のＴＳ−ｍｏｄｅでの音速
であり、電極膜側面部（膜厚）に付着反応した場合は膜厚部分に測定誤差が発生することになる。従って、リガンドの形成表面と凹部底面の表面と同一（平坦になる）にすることで、本願発明の主眼でもある、計測精度の向上と流路６から試料を注入した場合の試料の流れが更に円滑になることで、測定効率を高めることができる。 dF = − (dMF 2 ) / (Aρk f ) (1)
dF 2 : Frequency change dM: Mass change F: Excitation frequency A: Electrode area ρ: Quartz density k f : Sound velocity in TS-mode of quartz, and when it adheres and reacts on the side surface (film thickness) of the electrode film A measurement error occurs in the film thickness portion. Therefore, by making the surface on which the ligand is formed and the surface of the bottom surface of the concave portion the same (flat), the improvement in measurement accuracy and the flow of the sample when the sample is injected from the flow path 6 are the main features of the present invention. Measurement efficiency can be improved by becoming smooth.

しかしながら、凹部底面に形成するリガンドの全体高さは僅かであるために、図１で示す形態であっても、図２で示す形態であっても同様に試料の流れの円滑性と、リアルタイムで検体を測定できることには変わりは無い。   However, since the overall height of the ligand formed on the bottom surface of the recess is very small, the sample flow is smooth and real-time in the form shown in FIG. 1 and the form shown in FIG. There is no change in the ability to measure the specimen.

そして、図３は図１の形態を例にした場合の断面図である。図３に示すように実際に微少質量検出チップとして動作するときには、電気的特性と気密性を維持するためにスルーホ−ル４をシール材で埋めた構造となっている。ここではシール材５としては金シリコン合金を用いているが、イオン化傾向の小さな材料を使用すれば、金シリコン合金に限るものでは無い。   FIG. 3 is a cross-sectional view when the embodiment of FIG. 1 is taken as an example. As shown in FIG. 3, when actually operating as a minute mass detection chip, the through-hole 4 is filled with a sealing material in order to maintain electrical characteristics and airtightness. Here, a gold-silicon alloy is used as the sealing material 5, but the material is not limited to a gold-silicon alloy if a material with a small ionization tendency is used.

なお、本願発明の微少質量検出チップでは、所定の間隔でそれぞれ分離した複数の凹部を備えた圧電単結晶基板の凹部の表裏に励振電極を形成し、一方面の電極を反応電極としそれぞれの反応側電極上に反応状態を捉えるリガンドを形成し、形成された励振電極の引き出し電極の一方を、前記チップの主面にスルーホ−ル４を形成し、スルーホールを介して反対面に引き出したことを特徴として記述しているが、圧電基板上に形成する凹部に試験薬を貯めるために、凹部側に蓋体を被せた構造と、試料（検体）の量の節約、汚染防止などの効果がある。   In the minute mass detection chip of the present invention, excitation electrodes are formed on the front and back of the recesses of the piezoelectric single crystal substrate each having a plurality of recesses separated at predetermined intervals, and each electrode is used as a reaction electrode. A ligand that captures the reaction state was formed on the side electrode, and one of the extraction electrodes of the formed excitation electrode was formed on the main surface of the chip with a through hole 4 and extracted to the opposite surface through the through hole. In order to store the test drug in the recess formed on the piezoelectric substrate, the structure with the lid on the recess side, the amount of sample (specimen) saved, and the effect of preventing contamination, etc. is there.

図４に示す模式図は蓋体を割愛した図で、本願発明の特徴である流路６の構成と、凹部内の流路６の入口と出口との形態を説明する図である。図４では矢印で示す方向で溶液を流すことを表示したものである。この形態によりその微少質量検出チップにおいては、個々の微少質量検出チップの前記圧電単結晶基板に形成する溝部の流路の凹部内側の一方には、２稜線に略１２０°拡がる流路６があり、他方の流路にも２稜線に略１２０°拡がる流路６により他方の前記流路に連結した微少質量検出チップが連続した形態を有することができ、リアルタイムに反応状態を観測することが可能となる。なお、１２０°に拡がる流路６のにより、その周辺部の試料の溜まりを防止することができる。   The schematic diagram shown in FIG. 4 is a diagram omitting the lid, and is a diagram for explaining the configuration of the flow path 6 and the forms of the inlet and outlet of the flow path 6 in the recess, which are features of the present invention. In FIG. 4, the flow of the solution in the direction indicated by the arrow is displayed. According to this configuration, in the minute mass detection chip, there is a flow channel 6 that extends approximately 120 ° on two ridge lines on one of the inner sides of the groove of the groove portion formed in the piezoelectric single crystal substrate of each small mass detection chip. In addition, the other flow path can have a continuous form of a minute mass detection chip connected to the other flow path by a flow path 6 extending approximately 120 ° on two ridge lines, and the reaction state can be observed in real time. It becomes. In addition, accumulation of the sample in the peripheral part can be prevented by the flow path 6 extending to 120 °.

そして同時に、微少質量検出チップ同士を連続して配置し、各々のチップを流路６により接続することで、異なる特有の塩基配列のＤＮＡを用意しておき、同一検体ＤＮＡ溶液を流路６を介して流すことで、複数の反応結果を得ることができる。   At the same time, minute mass detection chips are continuously arranged, and each chip is connected by a flow path 6 to prepare DNAs having different specific base sequences. A plurality of reaction results can be obtained.

本発明の一実施例を示す斜視図（ａ）と部分断面図（ｂ）である。It is the perspective view (a) and partial sectional view (b) which show one Example of this invention. 本発明の他の実施例を示す斜視図（ａ）と部分断面図（ｂ）である。It is the perspective view (a) and partial sectional view (b) which show the other Example of this invention. 本発明の微少質量検出チップを個別で見たときの、スルーホール部分をシールで塞いだ概念図を示す模式図である。It is a schematic diagram which shows the conceptual diagram which closed the through-hole part with the seal | sticker when the minute mass detection chip | tip of this invention is seen separately. 本願発明の要部を説明する拡大図である。It is an enlarged view explaining the principal part of this invention.

符号の説明Explanation of symbols

１ 水晶基板
２ 凹部
３ 金薄膜（引き出し電極）
４ スルーホール
５ シール材
６ 流路 1 Crystal substrate 2 Recess 3 Gold thin film (lead electrode)
4 Through hole 5 Sealing material 6 Flow path

Claims (6)

所定の間隔でそれぞれ分離した複数の凹部を備えた圧電単結晶基板の凹部の表裏に励振電極を形成し、一方面の電極を反応電極としそれぞれの該反応側電極上に反応状態を捉えるリガンドを形成した微少質量検出チップにおいて、
前記凹部を形成する圧電単結晶基板の一部に溝部の流路を形成し、前記流路から試薬を注入することを特徴とする微少質量検出チップ。 Excitation electrodes are formed on the front and back sides of the concave portion of the piezoelectric single crystal substrate having a plurality of concave portions separated at predetermined intervals, and a ligand that captures the reaction state on each reaction side electrode with the electrode on one side as a reaction electrode. In the formed micro mass detection chip,
A micro-mass detection chip, wherein a channel of a groove is formed in a part of a piezoelectric single crystal substrate forming the recess, and a reagent is injected from the channel. 請求項１記載の微少質量検出チップにおいて、個々の微少質量検出チップの前記圧電単結晶基板に形成する流路凹部内側の一方には、２稜線に略１２０°拡がる流路があり、他方の溝部の流路にも２稜線に略１２０°拡がる流路により他方の流路に連結した微少質量検出チップが連続していることを特徴とする微少質量検出チップ。 2. The micro-mass detection chip according to claim 1, wherein one of the micro-mass detection chips inside the channel recess formed in the piezoelectric single crystal substrate has a channel extending approximately 120 ° along two ridge lines, and the other groove portion. A minute mass detection chip connected to the other channel by a channel extending approximately 120 ° along two ridges is also connected to the other channel. 請求項１記載の微少質量検出チップにおいて、形成された励振電極の引き出し電極の一方を、前記チップの主面にスルーホ−ルを形成し前記スルーホールを介して対面側に引き出したことを特徴とする微少質量検出チップ。 2. The micro-mass detection chip according to claim 1, wherein one of the lead electrodes of the formed excitation electrode is formed on the main surface of the chip and a through hole is formed on the opposite side through the through hole. A small mass detection chip. 請求項３記載の微少質量検出チップにおいて、電気的特性・気密性を維持するため、スルーホ−ルをシール材で埋めたことを特徴とする微少質量検出チップ。 4. The micro mass detection chip according to claim 3, wherein the through hole is filled with a sealing material in order to maintain electrical characteristics and airtightness. 請求項１ないし請求項４に記載の微小質量検出チップにおいて、凹部の反応膜下の基板部位を反応膜の厚みと同等にまでへこませて加工し、反応側凹部底面を平らにしたことを特徴とする微少質量検出チップ。 5. The micro mass detection chip according to claim 1, wherein the substrate portion under the reaction film in the recess is dented to the same thickness as the reaction film, and the bottom of the reaction side recess is flattened. A very small mass detection chip. 請求項１ないし請求項５に記載の微少質量検出チップにおいて、複数の励振電極（反応側電極）上に、それぞれ異なる塩基配列のＤＮＡ、或いは、ＲＮＡ断片が固定されたことを特徴とする微少質量検出チップ。 6. The minute mass detection chip according to claim 1, wherein DNAs or RNA fragments having different base sequences are immobilized on a plurality of excitation electrodes (reaction side electrodes). Detection chip.
JP2005051798A 2005-02-25 2005-02-25 Micro mass detection chip Expired - Fee Related JP4597710B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005051798A JP4597710B2 (en) 2005-02-25 2005-02-25 Micro mass detection chip

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2005051798A JP4597710B2 (en) 2005-02-25 2005-02-25 Micro mass detection chip

Publications (2)

Publication Number Publication Date
JP2006234686A true JP2006234686A (en) 2006-09-07
JP4597710B2 JP4597710B2 (en) 2010-12-15

Family

ID=37042487

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2005051798A Expired - Fee Related JP4597710B2 (en) 2005-02-25 2005-02-25 Micro mass detection chip

Country Status (1)

Country Link
JP (1) JP4597710B2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014240809A (en) * 2013-06-12 2014-12-25 日本電波工業株式会社 Sensing device

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06221980A (en) * 1992-10-16 1994-08-12 Nippon Steel Corp Measuring apparatus for material in liquid
JPH0875628A (en) * 1994-09-09 1996-03-22 Nippon Steel Corp Flow cell for continuous measurement of adsorbate amount in fluid
JP2000283905A (en) * 1999-03-30 2000-10-13 Noboru Koyama Multichannel qcm sensor device
JP2003155300A (en) * 2001-11-15 2003-05-27 Protein Crystal:Kk Protein chip and its use to chemical reaction detection
JP2003287538A (en) * 2002-01-28 2003-10-10 Kinseki Ltd Dna chip and dna testing method
JP2003307480A (en) * 2002-04-17 2003-10-31 Canon Inc Apparatus and system for measuring smallmass and method of taking out electrodes
JP2004061253A (en) * 2002-07-26 2004-02-26 Seiko Epson Corp Dispenser, dispenser array, method for manufacturing the dispenser, inspection apparatus, inspection method, and biochip
JP2004510145A (en) * 2000-09-20 2004-04-02 モレキュラー・リフレクションズ Microfabricated ultrasound arrays for use as resonance sensors
JP2004226405A (en) * 2003-01-24 2004-08-12 Lg Electron Inc Material sensing sensor and module using thin-film bulk accoustic resonator
JP2004264254A (en) * 2003-03-04 2004-09-24 Seiko Epson Corp Mass measuring chip and masses measuring device
JP2004309462A (en) * 2003-02-26 2004-11-04 Toshiba Corp Chip, analyzer and method for quantitative analysis of nucleic acid concentration
JP2005033293A (en) * 2003-07-08 2005-02-03 Toyo Commun Equip Co Ltd Piezoelectric device
JP2005051408A (en) * 2003-07-31 2005-02-24 Seiko Epson Corp Piezoelectric device and its manufacturing method, mobile telephone device using piezoelectric device, and electronic equipment using piezoelectric device

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06221980A (en) * 1992-10-16 1994-08-12 Nippon Steel Corp Measuring apparatus for material in liquid
JPH0875628A (en) * 1994-09-09 1996-03-22 Nippon Steel Corp Flow cell for continuous measurement of adsorbate amount in fluid
JP2000283905A (en) * 1999-03-30 2000-10-13 Noboru Koyama Multichannel qcm sensor device
JP2004510145A (en) * 2000-09-20 2004-04-02 モレキュラー・リフレクションズ Microfabricated ultrasound arrays for use as resonance sensors
JP2003155300A (en) * 2001-11-15 2003-05-27 Protein Crystal:Kk Protein chip and its use to chemical reaction detection
JP2003287538A (en) * 2002-01-28 2003-10-10 Kinseki Ltd Dna chip and dna testing method
JP2003307480A (en) * 2002-04-17 2003-10-31 Canon Inc Apparatus and system for measuring smallmass and method of taking out electrodes
JP2004061253A (en) * 2002-07-26 2004-02-26 Seiko Epson Corp Dispenser, dispenser array, method for manufacturing the dispenser, inspection apparatus, inspection method, and biochip
JP2004226405A (en) * 2003-01-24 2004-08-12 Lg Electron Inc Material sensing sensor and module using thin-film bulk accoustic resonator
JP2004309462A (en) * 2003-02-26 2004-11-04 Toshiba Corp Chip, analyzer and method for quantitative analysis of nucleic acid concentration
JP2004264254A (en) * 2003-03-04 2004-09-24 Seiko Epson Corp Mass measuring chip and masses measuring device
JP2005033293A (en) * 2003-07-08 2005-02-03 Toyo Commun Equip Co Ltd Piezoelectric device
JP2005051408A (en) * 2003-07-31 2005-02-24 Seiko Epson Corp Piezoelectric device and its manufacturing method, mobile telephone device using piezoelectric device, and electronic equipment using piezoelectric device

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014240809A (en) * 2013-06-12 2014-12-25 日本電波工業株式会社 Sensing device

Also Published As

Publication number Publication date
JP4597710B2 (en) 2010-12-15

Similar Documents

Publication Publication Date Title
Patel et al. Biosensors in health care: the milestones achieved in their development towards lab-on-chip-analysis
JP3693572B2 (en) Nanoelectrode array
JP2022058772A (en) Devices and methods for sample analysis
JP4775262B2 (en) Sensor unit, reaction field cell unit and analyzer
JP4763066B2 (en) Apparatus and method for analyte measurement and immunoassay
ES2691647T3 (en) Method and apparatus for detecting targets using viruses attached to electrodes
CN100378451C (en) Microelectronic detector on chip
Rahong et al. Recent developments in nanowires for bio-applications from molecular to cellular levels
US20070238112A1 (en) Microfluidic and nanofluidic electronic devices for detecting changes in capacitance of fluids and methods of using
US20050058990A1 (en) Biochip devices for ion transport measurement, methods of manufacture, and methods of use
JP2018511805A (en) Devices and methods for sample analysis
WO2005001018A1 (en) Pharmacological measuring device and system and well container used therefor
US20140231274A1 (en) Single molecule detection method and single molecule detection apparatus for biological molecule, and disease marker testing apparatus
AU2019261360A1 (en) New apparatus and methods for disease detection
WO2011102885A1 (en) Sensing device and related methods
US20180364188A1 (en) Modular chemiresistive sensor for in vitro diagnostic and gas sensing applications
Lemay et al. Single-entity electrochemistry for digital biosensing at ultralow concentrations
Guan et al. Glass capillary-based nanopores for single molecule/single cell detection
JP4597710B2 (en) Micro mass detection chip
US20100234234A1 (en) Methods and Devices for Detecting Structural Changes in a Molecule Measuring Electrochemical Impedance
JP4597711B2 (en) Micro mass detection chip
JP2006234685A (en) Chip for detecting small mass
JP2006208284A (en) Trace quantity mass sensor mounted chip and trace quantity mass analyzing system, and analyzing method of the trace quantity mass sensor mounted chip
JP4141442B2 (en) Sensor and protein detection device
US20220002795A1 (en) Nano-pen sequencing: an integrated nanotube and tunnel gap platform for polymer sequencing

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20080221

A977 Report on retrieval

Free format text: JAPANESE INTERMEDIATE CODE: A971007

Effective date: 20100527

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20100608

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20100806

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20100831

A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20100922

R150 Certificate of patent or registration of utility model

Ref document number: 4597710

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150

Free format text: JAPANESE INTERMEDIATE CODE: R150

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20131001

Year of fee payment: 3

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20131001

Year of fee payment: 3

S533 Written request for registration of change of name

Free format text: JAPANESE INTERMEDIATE CODE: R313533

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20131001

Year of fee payment: 3

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

S531 Written request for registration of change of domicile

Free format text: JAPANESE INTERMEDIATE CODE: R313531

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

S111 Request for change of ownership or part of ownership

Free format text: JAPANESE INTERMEDIATE CODE: R313111

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

LAPS Cancellation because of no payment of annual fees