JP2006275798A - Method of immobilizing cell onto electrode of piezoelectric element, method of measuring cell volume, and measuring instrument for cell volume - Google Patents
Method of immobilizing cell onto electrode of piezoelectric element, method of measuring cell volume, and measuring instrument for cell volume Download PDFInfo
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本発明は、圧電素子の電極への細胞の固定化方法、並びに、細胞の物性、詳細には、形状変化（容積変化、リガンド−レセプター間の相互作用、細胞間相互作用）をリアルタイムで測定する方法に関する。 The present invention is a method for immobilizing cells on an electrode of a piezoelectric element, and measuring physical properties of cells, specifically, shape changes (volume change, ligand-receptor interaction, cell-cell interaction) in real time. Regarding the method.
従来、リガンドとレセプター間の相互作用を測定する方法としては、蛍光物質などのリガンド標識体を用いた細胞染色、細胞膜分画を用いた固定アッセイ、生体内標識（Ｃ１４等）を用いた免疫沈降法などの方法がある。
リガンドとレセプター間の相互作用を解析する手段のほとんどは、精製した分子同士を用いた測定系であり、生体内で引き起こされている相互作用を完全に再現されているか否かが不明であるという問題があった。また、蛍光標識を用いた従来の方法では、標識物質を用いるために、本来の特性とは異なる特性を示してしまうという問題があった。また、細胞膜画分からレセプター等の機能分子の機能を維持したまま精製することは難しいし、得られた分子が本来の機能を維持することは難しいという問題があった。
この問題を解決するために、特許文献１には、水晶発振子を使用し、電極上に生細胞を、チオアルキルカルボン酸又はジチオジアルキルカルボン酸と、１−（３−ジメチルアミノプロピル）−３−エチルカルボジイミドと、Ｎ−ヒドロキシスクシンイミドとを使用すること等して固定化することが提案されている。
しかしながら、特許文献１に提案されるような方法では、細胞を固定化する際に細胞にダメージを与えるおそれがあった。
Conventionally, methods for measuring the interaction between a ligand and a receptor include cell staining using a ligand-labeled substance such as a fluorescent substance, fixed assay using a cell membrane fraction, and immunoprecipitation using an in vivo label (C14 etc.). There are methods such as law.
Most of the means for analyzing the interaction between the ligand and the receptor is a measurement system using purified molecules, and it is unclear whether the interaction caused in vivo is completely reproduced. There was a problem. In addition, the conventional method using a fluorescent label has a problem that a characteristic different from the original characteristic is exhibited because a labeling substance is used. In addition, it is difficult to purify the cell membrane fraction while maintaining the function of a functional molecule such as a receptor, and it is difficult for the obtained molecule to maintain its original function.
In order to solve this problem, Patent Document 1 discloses that a crystal oscillator is used, a living cell is placed on an electrode, thioalkylcarboxylic acid or dithiodialkylcarboxylic acid, and 1- (3-dimethylaminopropyl) -3. It has been proposed to immobilize by using ethyl carbodiimide and N-hydroxysuccinimide.
However, in the method proposed in Patent Document 1, there is a possibility that the cells are damaged when the cells are immobilized.
このような状況下において、細胞の環境変化やアポトーシス等に伴う形状変化を測定するために、顕微鏡観察や細胞膜の電位差を測定することが行われている。
しかしながら、従来の方法では、先にも述べたように、固定化の際に細胞にダメージを与えるおそれがあり、また、測定中に細胞の特性が変化することがあり、本来の細胞の特性について測定することができないという問題があった。更に、顕微鏡観察の方法では、細胞の形状変化は顕微鏡の画像から算出しているため、微少な変化量や形状変化が開始した直後に生じる変化を測定することが困難であった。また、膜電位を測定する方法では、複数の細胞にバラツキがあるため、正確な形状測定ができないという問題があった。
Under such circumstances, in order to measure changes in the cell environment and changes in shape associated with apoptosis, etc., microscopic observation and measurement of the potential difference of the cell membrane are performed.
However, in the conventional method, as described above, there is a risk of damaging cells during immobilization, and the characteristics of cells may change during measurement. There was a problem that it could not be measured. Furthermore, in the microscopic observation method, since the cell shape change is calculated from a microscope image, it is difficult to measure a slight change amount or a change that occurs immediately after the shape change starts. In addition, the method for measuring the membrane potential has a problem in that accurate shape measurement cannot be performed due to variations in a plurality of cells.
そこで、本発明は、細胞を傷めることなく圧電素子の電極上に固定化することができる方法と、細胞本来の特性に基づいた細胞の形状変化を電極上に固定化された細胞の重量変化を測定することによりリアルタイムに測定することが可能な方法とを提供することを目的とする。 Therefore, the present invention provides a method capable of immobilizing cells on the electrode of the piezoelectric element without damaging the cells, and a change in cell weight based on the original characteristics of the cells. It is an object to provide a method capable of measuring in real time by measuring.
上記課題を解決するために、本発明者等は鋭意検討の結果、下記の通り解決手段を見いだした。
即ち、本発明は、請求項１に記載の通り、圧電素子の検出部表面において、細胞を培養することにより、前記細胞を前記検出部表面に固定化することを特徴とする。
また、請求項２に記載の本発明は、請求項１に記載の圧電素子への細胞の固定化方法において、前記細胞は、培養細胞であることを特徴とする。
また、請求項３に記載の本発明は、請求項１又は２に記載の圧電素子への細胞の固定化方法において、前記圧電素子は、水晶振動子であり、前記検出部を電極により構成し、前記電極を、金又は白金としたことを特徴とする。
また、請求項４に記載の本発明は、請求項１に記載の圧電素子への細胞の固定化方法において、前記細胞は、浮遊細胞であり、前記検出部表面に細胞粘着剤層を設け、前記細胞粘着剤層上において前記浮遊細胞を培養することにより、前記浮遊細胞を前記細胞粘着剤層に固定化することを特徴とする。
本発明の細胞容積の測定方法は、請求項５に記載の通り、細胞が固定化された圧電素子を発振させ、前記圧電素子の物理的特性の変化に基づいて、前記細胞の容積の変化を測定することを特徴とする。
また、請求項６に記載の本発明は、請求項１乃至４のいずれかに記載の方法により前記細胞が固定化された前記圧電素子を発振させ、前記圧電素子の物理的特性の変化に基づいて、前記細胞容積の変化を測定することを特徴とする。
本発明の細胞容積の測定装置は、請求項７に記載の通り、請求項１乃至４のいずれかに記載の方法により前記細胞が固定化された圧電素子を備えることができるようにしたことを特徴とする。
In order to solve the above-mentioned problems, the present inventors have found a solving means as follows as a result of intensive studies.
That is, the present invention is characterized in that, as described in claim 1, the cells are immobilized on the surface of the detection unit by culturing the cells on the surface of the detection unit of the piezoelectric element.
According to a second aspect of the present invention, in the method for immobilizing a cell on the piezoelectric element according to the first aspect, the cell is a cultured cell.
According to a third aspect of the present invention, there is provided the method for immobilizing a cell on the piezoelectric element according to the first or second aspect, wherein the piezoelectric element is a crystal resonator, and the detection unit is configured by an electrode. The electrode is made of gold or platinum.
Further, the present invention according to claim 4 is the method of immobilizing cells to the piezoelectric element according to claim 1, wherein the cells are floating cells, and a cell adhesive layer is provided on the surface of the detection unit, The floating cells are immobilized on the cell adhesive layer by culturing the floating cells on the cell adhesive layer.
According to the method for measuring a cell volume of the present invention, as described in claim 5, a piezoelectric element to which a cell is immobilized is oscillated, and a change in the volume of the cell is determined based on a change in physical characteristics of the piezoelectric element. It is characterized by measuring.
According to a sixth aspect of the present invention, the piezoelectric element to which the cells are immobilized is oscillated by the method according to any of the first to fourth aspects, and based on a change in physical characteristics of the piezoelectric element. Then, the change in the cell volume is measured.
According to a seventh aspect of the present invention, there is provided a device for measuring a cell volume, comprising the piezoelectric element on which the cells are immobilized by the method according to any one of the first to fourth aspects. Features.
本発明では、圧電素子を構成する電極上で細胞を培養して固定化することにより、細胞にダメージを与えることなく、その特性を損なわない状態で固定化することができる。
また、本発明によれば、細胞の重量変化を振動数等の物理的特性の変化に基づいて測定することができるので、微少な細胞の形状変化であっても数値化して測定することができる。また、細胞の形状変化が開始した直後であってもリアルタイムに測定することができる。更に、圧電素子の電極上に固定化した細胞全ての形状の変化量が振動数等の物理的特性の変化に基づいて測定できるので、固体差間の相違の影響はなく、平均的な変化量として測定することができる。
また、上記電極上で細胞を培養して固定化するようにした場合には、細胞に標識物質等を導入する必要がなく、より自然な状態で測定を行えるため、細胞本来の特性に基づいた細胞の形状変化が測定できる。
In the present invention, by culturing and immobilizing cells on the electrodes constituting the piezoelectric element, the cells can be immobilized without damaging the characteristics without damaging the cells.
In addition, according to the present invention, a change in the weight of a cell can be measured based on a change in physical characteristics such as a frequency, so that even a slight change in the shape of a cell can be quantified and measured. . In addition, measurement can be performed in real time even immediately after the start of cell shape change. In addition, the amount of change in the shape of all cells immobilized on the electrodes of the piezoelectric element can be measured based on changes in physical properties such as vibration frequency, so there is no difference between individual differences and the average amount of change. Can be measured as
In addition, when cells are cultured and immobilized on the electrode, it is not necessary to introduce a labeling substance or the like into the cells, and measurement can be performed in a more natural state. Changes in cell shape can be measured.
上記の通り、本発明の細胞の固定化方法は、圧電素子の電極上において、細胞を培養することにより固定化するものである。
本発明に使用することができる圧電素子としては、水晶振動子や表面弾性波素子等を使用することができる。
前記弾性波素子としては、圧電基板内又は圧電基板表面に弾性波を励起することができるものであれば特に制限はないが、ラブ波デバイス、ＳＨ−ＳＡＷデバイス、ＳＴＷデバイス、ＦＰＷデバイス又はＡＰＭデバイスを使用することが好ましい。
前記ラブ波デバイスは、圧電材料であるＳＴカット水晶、ＬｉＴａＯ3等からなる基板にＡｕ、Ａｌ、Ｃｒ等の金属膜から構成されるＩＤＴを設け、これらの上から、前記基板の横波の伝達速度より遅い速度を有する材質（ＳｉＯ2、ポリマー等）を層状に設け、波の伝播方向に垂直で、基板表面に平行な横波成分の表面波（ラブ波）を励起することができる構造をしたものである。
前記ＳＨ−ＳＡＷデバイスは、圧電材料であるＡＴカット水晶基板等にＩＤＴのグレーティング（溝）を設けたものであり、基板を伝播するＳＳＢＷ（Surface Skimming Bulk Waves）をグレーティングにより基板表面にトラップし、表面横波（Surface Transverse Waves）を励起することができる構造をしたものである。
前記ＦＰＷデバイスは、圧電材料基板やＺｎＯ膜等の圧電材料薄膜上にＩＤＴを設けたものであり、波の変位が波の伝播方向と基板に垂直方向の成分からなるラム波と呼ばれる板波を励起する構造をしたものである。
前記ＡＰＭデバイスは、圧電材料であるＳＴカット水晶基板上にＩＤＴを設けたものであり、基板表面に沿って基板に平行伝播するＳＨタイプの板波を励起する構造をしたものである。
尚、上記水晶振動子における検出部は、水晶板の表面に設けられた電極であり、表面弾性波素子における検出部は、弾性波が伝播する部位をいうものとする。また、前記水晶振動子の電極は、金又は白金から構成することが好ましい。細胞の固定化が、他の金属に比べて確実に行えることが確認できているためである。
As described above, the cell immobilization method of the present invention immobilizes cells by culturing them on the electrodes of the piezoelectric element.
As a piezoelectric element that can be used in the present invention, a crystal resonator, a surface acoustic wave element, or the like can be used.
The acoustic wave element is not particularly limited as long as it can excite an acoustic wave in the piezoelectric substrate or on the surface of the piezoelectric substrate, but is not limited to a Love wave device, SH-SAW device, STW device, FPW device, or APM device. Is preferably used.
In the Love wave device, an IDT composed of a metal film such as Au, Al, Cr, etc. is provided on a substrate made of ST cut quartz, LiTaO 3 or the like which is a piezoelectric material. A material with a slower speed (SiO 2 , polymer, etc.) is provided in layers, and a structure that can excite a surface wave (love wave) of a transverse wave component that is perpendicular to the wave propagation direction and parallel to the substrate surface It is.
The SH-SAW device has an IDT grating (groove) provided on an AT-cut quartz substrate, which is a piezoelectric material, and traps SSBW (Surface Skimming Bulk Waves) propagating through the substrate on the substrate surface by the grating. It has a structure that can excite surface transverse waves.
In the FPW device, an IDT is provided on a piezoelectric material substrate or a piezoelectric material thin film such as a ZnO film, and a plate wave called a Lamb wave in which a wave displacement is composed of a component in a wave propagation direction and a direction perpendicular to the substrate. Excited structure.
The APM device has an IDT provided on an ST-cut quartz substrate, which is a piezoelectric material, and has a structure that excites SH type plate waves that propagate in parallel to the substrate along the substrate surface.
The detection unit in the crystal resonator is an electrode provided on the surface of the crystal plate, and the detection unit in the surface acoustic wave element refers to a portion where the acoustic wave propagates. Moreover, it is preferable that the electrode of the crystal resonator is made of gold or platinum. This is because it has been confirmed that cells can be fixed more reliably than other metals.
上記した圧電素子の検出部における細胞の培養方法としては、特に制限はなく、通常の培養方法をにより行うことができる。例えば、電極上を滅菌してから、培養液として、DMEM（Dulbecco's modified eagle's medium）やOpti-MEM I等を滴下し、培養する物質を添加してインキュベータにより培養する方法がある。
また、検出部において培養できる細胞についても、特に制限するものでなく、HEK 293（ヒト胎児腎臓由来株）、HeLa （ヒト上皮細胞）やBHK21 (シリアンハムスター繊維芽細胞）等の培養細胞や赤血球等の浮遊細胞のいずれも培養することができる。但し、浮遊細胞の場合には、細胞粘着剤層上において培養する必要がある。尚、前記細胞粘着剤層としては、細胞を培養した際に、浮遊細胞が離れることなく固定化できるものであれば特に制限はなく、例えば、フィプロネクチン、コラーゲン等を使用することができる。
尚、本発明により培養して細胞を圧電素子に固定化する場合には、細胞は、自らが形成したタンパク質（接着剤）に覆われているため、表面は滑らかではないのに対して、培養器で培養して細胞を圧電素子に固定化した場合には、培養器で培養した細胞を回収する際に細胞表面をトリプシン（タンパク質分解剤）等により処理するために表面の接着剤がとれ、細胞表面が滑らかな形状となる。この相違は、顕微鏡等の表面を観察することにより認識することができる。
There is no restriction | limiting in particular as a cell culture method in the detection part of an above-described piezoelectric element, It can carry out by a normal culture method. For example, after sterilizing the electrode, there is a method in which DMEM (Dulbecco's modified eagle's medium), Opti-MEM I, or the like is dropped as a culture solution, and a substance to be cultured is added and cultured in an incubator.
Also, the cells that can be cultured in the detection section are not particularly limited, and cultured cells such as HEK 293 (human embryonic kidney-derived strain), HeLa (human epithelial cells) and BHK21 (Syrian hamster fibroblasts), red blood cells, etc. Any of the floating cells can be cultured. However, in the case of floating cells, it is necessary to culture on the cell adhesive layer. The cell adhesive layer is not particularly limited as long as the cells can be immobilized without leaving floating cells when cells are cultured. For example, fipronectin, collagen and the like can be used.
In the case of immobilizing cells on a piezoelectric element by culturing according to the present invention, since the cells are covered with the protein (adhesive) formed by themselves, the surface is not smooth. When the cells are fixed to the piezoelectric element by culturing in a vessel, the surface adhesive is removed to treat the cell surface with trypsin (proteolytic agent) or the like when the cells cultured in the incubator are collected, The cell surface has a smooth shape. This difference can be recognized by observing the surface of a microscope or the like.
次に、本発明の細胞容積の測定方法について説明する。
本発明の測定方法は、細胞が固定化された圧電素子を発振させ、前記圧電素子の物理的特性の変化に基づいて、前記細胞の容積の変化を測定するものである。
圧電素子の水晶振動子や表面弾性波素子を発振又は表面弾性波を励起して、検出部における周波数や弾性波の伝播速度等の物理的特性の変化を測定する方法としては、公知の方法を利用することができる。
また、本発明の測定方法に、上記本発明により培養することにより圧電素子に固定化された細胞を使用することが好ましい。本来の特性を損なわない細胞の容積を測定することができるからである。
Next, the cell volume measuring method of the present invention will be described.
The measurement method of the present invention oscillates a piezoelectric element in which cells are immobilized, and measures a change in the volume of the cell based on a change in physical characteristics of the piezoelectric element.
As a method of measuring changes in physical characteristics such as frequency and acoustic wave propagation speed in the detection unit by oscillating a piezoelectric vibrator or surface acoustic wave element of a piezoelectric element or exciting surface acoustic waves, a known method is used. Can be used.
Moreover, it is preferable to use the cell fixed to the piezoelectric element by culture | cultivating by the said this invention for the measuring method of this invention. This is because the volume of cells that does not impair the original characteristics can be measured.
次に、本発明の細胞容積の測定装置について説明する。
本発明の細胞容積の測定装置は、上記本発明により培養することにより圧電素子に細胞が固定化されたものを備えることができるようにしたものである。
以下、図面を参照し、一実施の形態の細胞容積の測定装置について説明するが、本発明は、この実施の形態により制限されるものではない。
図５に示されるものは、本発明の一実施の形態である細胞容積の測定装置１を示すものである。
この細胞容積の測定装置１は、センサー部２と、ネットワークアナライザ３と、コンピュータ４とを備えている。センサー部２とネットワークアナライザ３及びネットワークアナライザ３とコンピュータ４とは、それぞれケーブル５と６を介して接続されている。また、センサー部２は、図５では図示しないが水晶振動子を備えている。
Next, the cell volume measuring apparatus of the present invention will be described.
The cell volume measuring apparatus according to the present invention can be provided with a cell in which cells are immobilized on a piezoelectric element by culturing according to the present invention.
Hereinafter, a cell volume measuring apparatus according to an embodiment will be described with reference to the drawings. However, the present invention is not limited to the embodiment.
FIG. 5 shows a cell volume measuring apparatus 1 according to an embodiment of the present invention.
The cell volume measuring device 1 includes a sensor unit 2, a network analyzer 3, and a computer 4. The sensor unit 2 and the network analyzer 3 and the network analyzer 3 and the computer 4 are connected via cables 5 and 6, respectively. In addition, the sensor unit 2 includes a crystal resonator (not shown in FIG. 5).
測定装置１のセンサー部２に設けられる水晶振動子は、図６（ａ）、（ｂ）にその平面図と断面図とをそれぞれ示すように、円形状に形成された石英製の結晶板８の表面側と裏面側とにそれぞれ第一の金電極９と第二の金電極１０と備えている。尚、図示される金電極９ａ、１０ａは、円形状に構成され、それぞれの金電極９ａ、１０ａからリード線９ｂ、１０ｂが結晶板８の端部にまで延びている。裏面側の第二の金電極１０は、図６（ｂ）に示すように樹脂カバー１１により被覆されており、水晶振動子７を溶液中に浸漬した状態で、裏面側の第二の金電極１０が溶液に曝されず、発振できるように構成されている。他方、表面側の第一の金電極９の表面には、上記説明したように、第一の金電極９上に、培養することにより細胞１２が固定化されている。 The quartz crystal resonator provided in the sensor unit 2 of the measuring device 1 has a quartz crystal plate 8 formed in a circular shape as shown in FIGS. 6A and 6B in plan and sectional views, respectively. The first gold electrode 9 and the second gold electrode 10 are provided on the front surface side and the back surface side, respectively. The illustrated gold electrodes 9 a and 10 a are formed in a circular shape, and lead wires 9 b and 10 b extend from the respective gold electrodes 9 a and 10 a to the end of the crystal plate 8. The second gold electrode 10 on the back surface side is covered with a resin cover 11 as shown in FIG. 6 (b), and the second gold electrode on the back surface side is immersed in the crystal resonator 7 in the solution. 10 is configured not to be exposed to the solution and to oscillate. On the other hand, the cells 12 are immobilized on the surface of the first gold electrode 9 on the surface side by culturing on the first gold electrode 9 as described above.
また、装置１を構成するネットワークアナライザ３は、図７に示すように、信号供給回路１３と測定回路１４とを備えており、信号供給回路１３は、周波数を変化させながら交流の入力信号を出力することができるように構成されている。また、測定回路１４は、水晶振動子７の出力信号や、信号供給回路１３から出力される入力信号に基づいて、水晶振動子７の共振周波数や位相等の物理的特性を測定して、コンピュータ４に出力することができるように構成されている。
また、装置１を構成するコンピュータ４は、測定された水晶振動子７の周波数特性等の測定結果を表示することができるように構成されている。
The network analyzer 3 constituting the apparatus 1 includes a signal supply circuit 13 and a measurement circuit 14 as shown in FIG. 7, and the signal supply circuit 13 outputs an AC input signal while changing the frequency. It is configured to be able to. The measurement circuit 14 measures physical characteristics such as a resonance frequency and a phase of the crystal resonator 7 based on an output signal of the crystal resonator 7 and an input signal output from the signal supply circuit 13, and then calculates the computer. 4 is configured to be able to output to 4.
Further, the computer 4 constituting the apparatus 1 is configured to be able to display measurement results such as the measured frequency characteristics of the crystal resonator 7.
次に、上述した構成の細胞容積の測定装置１により、細胞容積を測定する手順について以下に説明する。
まず、図８に示すように、細胞等張液１６で満たされた円筒形状のセル１５底部に第一の電極９上に細胞１２が固定化された水晶振動子７を配置する。この状態で、コンピュータ４から制御信号を出力すると、ネットワークアナライザ３の信号供給回路１３からケーブル５を介してセンサー部２に所定の周波数で交流信号が供給され、水晶振動子７が共振周波数で発振する。
この状態で、セル１５内に低張液を添加すると、第１の金電極９上に固定化された細胞１２が膨らみ、水晶振動子７の共振周波数が低下し、ケーブル５、ネットワークアナライザ３、ケーブル６を介してコンピュータ４に出力される。
Next, the procedure for measuring the cell volume using the cell volume measuring apparatus 1 having the above-described configuration will be described below.
First, as shown in FIG. 8, the quartz crystal resonator 7 in which the cells 12 are immobilized on the first electrode 9 is disposed at the bottom of the cylindrical cell 15 filled with the cell isotonic solution 16. When a control signal is output from the computer 4 in this state, an AC signal is supplied from the signal supply circuit 13 of the network analyzer 3 to the sensor unit 2 via the cable 5 at a predetermined frequency, and the crystal resonator 7 oscillates at the resonance frequency. To do.
In this state, when a hypotonic solution is added into the cell 15, the cells 12 immobilized on the first gold electrode 9 swell, the resonance frequency of the crystal resonator 7 decreases, and the cable 5, the network analyzer 3, The data is output to the computer 4 via the cable 6.
尚、本実施の形態では、電気的特性を求める一例として、共振周波数を検出しているが、測定される周波数は、共振周波数に限られるものではなく、例えば、出力信号の位相と入力信号の位相との位相差を測定してもよい。特に、位相差が１８０°になる点（位相点）では、水晶振動子７は共振しているので、共振周波数を求める本実施の形態と同じことになる。また、上記以外にも、本出願人が先に提案した周波数（特願2003-120335又は特願2003-120370）を用いてもよい。粘性の影響がある場合は、この方がより正確な測定が可能となる。
また、周波数変化の測定に関しては、インピーダンスアナライザを使用する場合には、必ずしも、インピーダンスが最小となる点を測定することに限られない。例えば、本出願人が先に提案（特願2003−120335）した、振動子が直列共振状態にあるときのコンダクタンスの半分の大きさの半値コンダクタンスを与える半値周波数であって、前記直列共振状態を与える共振周波数に近く、且つ、共振周波数よりも大きな周波数である半値周波数を利用することもできる。また、同様に先に提案（特願2003−120370）した、振動子を直列状態に置く共振周波数と、前記振動子が共振状態にあるときのコンダクタンスの半分の大きさの半値コンダクタンスを与える第一、第二の半値周波数とで構成される三種類の周波数のうち、少なくとも二種類の周波数を利用することもできる。これにより、圧力波以外にも、バッファー液と粘性が異なる試料を使用した場合の粘性効果や温度変化による粘性効果の影響を受けることがないので正確な測定をすることが可能となる。
In this embodiment, the resonance frequency is detected as an example for obtaining the electrical characteristics. However, the measured frequency is not limited to the resonance frequency. For example, the phase of the output signal and the input signal You may measure the phase difference with a phase. In particular, at the point where the phase difference is 180 ° (phase point), the quartz crystal resonator 7 is resonating, which is the same as the present embodiment for obtaining the resonance frequency. In addition to the above, the frequency previously proposed by the present applicant (Japanese Patent Application 2003-120335 or Japanese Patent Application 2003-120370) may be used. In the case where there is an influence of viscosity, more accurate measurement is possible.
Further, regarding the measurement of the frequency change, when using an impedance analyzer, it is not necessarily limited to measuring the point where the impedance is minimum. For example, the half-frequency that has been proposed by the present applicant (Japanese Patent Application No. 2003-120335) to give a half-value conductance that is half the conductance when the vibrator is in a series resonance state, It is also possible to use a half-value frequency that is close to the resonance frequency to be applied and is larger than the resonance frequency. Similarly, the first proposed previously (Japanese Patent Application No. 2003-120370) provides a resonance frequency at which the vibrator is placed in series and a half-value conductance that is half the conductance when the vibrator is in the resonance state. Of the three types of frequencies constituted by the second half-value frequency, at least two types of frequencies can be used. As a result, in addition to the pressure wave, there is no influence of the viscosity effect when using a sample having a viscosity different from that of the buffer solution or the viscosity effect due to temperature change, and therefore, accurate measurement can be performed.
以下に、本発明の実施例について説明するが、本発明はこれらの実施例に限定されるものではない。 Examples of the present invention will be described below, but the present invention is not limited to these examples.
実施例１：犬上皮ガン細胞（以下、CSCC（Canine Squamous Cell Carcinoma）とする）の圧電素子の電極上への固定化と容積変化の測定
バイオセンサ用セルの底面に配置された板状の水晶振動子の電極にピランハ溶液（３０％過酸化水素水：濃硫酸＝１：３）を適量塗布し、５〜１０分間放置し、更に、同じ作業を行って電極部を洗浄した。前記セルに７０重量％のエタノールを噴霧した後、紫外線を数分間照射し、滅菌操作を行った。更に、前記滅菌処理されたセルに、培養液３２０μＬを入れ、この中に、CSCC１．３×１０4個を入れ、３７℃のＣＯ2インキュベータ内で３日間培養した。
培養後、アクリジンオレンジにて細胞を染色し、蛍光顕微鏡で観察した。その結果、圧電素子の電極部に細胞が固定化されたことが確認された。この細胞の蛍光顕微鏡写真による平面図を図１に示す。
次に、圧電素子の電極部に細胞を固定化したセルに、細胞等張液（２９５ｍＯｓＭ）を５００μＬを加え、水晶振動子装置（Affinix Q４：株式会社イニシアム製）にセットし、２７ＭＨｚで発振させた。前記水晶振動子の振動数が安定したところで、高張液１０μＬを添加し、浸透圧を変化させたときの振動数変化を維持測定した。その結果、細胞が収縮し、容積が小さくなったため、振動数は増加し、その後細胞の働きにより容積が通常に戻るに従って振動数が暫時低下した。この測定結果を図２に示す。
また、振動数が減少したところで、細胞等張液（２９５ｍＯｓＭ）を４００μＬ排水し、低張液を４００μＬ添加し、浸透圧を変化させたときの振動数変化を維持測定した。その結果、細胞は膨潤し、容積が大きくなったため、振動数は低下し、その後細胞の働きにより容積が通常に戻るに従って振動数が漸次増加した。この測定結果を図３に示す。
尚、図２及び図３中点線で示すグラフは、細胞が固定化されていないコントロールを示すグラフである。
Example 1: Immobilization of canine epithelial cancer cells (hereinafter referred to as CSCC (Canine Squamous Cell Carcinoma)) on an electrode of a piezoelectric element and measurement of volume change A plate-like crystal placed on the bottom of a biosensor cell An appropriate amount of piranha solution (30% aqueous hydrogen peroxide: concentrated sulfuric acid = 1: 3) was applied to the electrodes of the vibrator, left for 5 to 10 minutes, and the same operation was performed to clean the electrode part. The cell was sprayed with 70% by weight of ethanol and then irradiated with ultraviolet rays for several minutes for sterilization. Further, 320 μL of the culture solution was placed in the sterilized cell, and 1.3 × 10 4 CSCCs were placed therein, and cultured in a CO 2 incubator at 37 ° C. for 3 days.
After incubation, the cells were stained with acridine orange and observed with a fluorescence microscope. As a result, it was confirmed that the cells were immobilized on the electrode portion of the piezoelectric element. A plan view of this cell by a fluorescence micrograph is shown in FIG.
Next, 500 μL of a cell isotonic solution (295 mOsM) is added to a cell in which cells are fixed to the electrode part of the piezoelectric element, and the cell is set in a crystal resonator device (Affinix Q4: manufactured by Initium Co., Ltd.) and oscillated at 27 MHz. It was. When the frequency of the crystal resonator was stabilized, 10 μL of hypertonic solution was added, and the change in frequency when the osmotic pressure was changed was maintained and measured. As a result, since the cells contracted and the volume became smaller, the frequency increased, and then the frequency decreased for a while as the volume returned to normal due to the action of the cells. The measurement results are shown in FIG.
When the frequency decreased, 400 μL of cell isotonic solution (295 mOsM) was drained, 400 μL of hypotonic solution was added, and the change in frequency when the osmotic pressure was changed was maintained and measured. As a result, since the cells swelled and the volume increased, the frequency decreased, and then the frequency gradually increased as the volume returned to normal due to the action of the cells. The measurement results are shown in FIG.
In addition, the graph shown with the dotted line in FIG.2 and FIG.3 is a graph which shows the control by which the cell is not fix | immobilized.
実施例２：CSCCのコラーゲン粘着材層を使用した圧電素子電極上への固定化と容積変化の測定
バイオセンサ用セルの圧電素子の電極にピランハ溶液（３０％ 過酸化水素水：濃硫酸＝１：３）を適量塗布し、５〜１０分間放置し、更に、同じ作業を行い電極部を洗浄した。次に、コラーゲン溶液（新田ゼラチン社製 Cellmatrix TypeI−C）を１０倍に希釈し、圧電素子の電極上に薄く塗布し、乾燥させた後、紫外線を約１０分間照射し滅菌した。
前記セルを培養液で洗浄し、測定バッファー（Ｔｒｉｓ／ＭＯＰＳ：１０ｍＭ、ＮａＣｌ：１３５ｍＭ、ＫＣｌ：５ｍＭ、glucose：５ｍＭ）を５００μＬ添加した。そして、前記セルを、水晶振動子装置（Affinix Q４：株式会社イニシアム製）にセットし、２７ＭＨｚで発振させ、振動数を測定し、その結果を図４に示す。
前記セルから、測定バッファーを除去し、７０％エタノールを噴霧した後、紫外線を約１０分間照射し、滅菌した。滅菌したセルに培養液３２０μＬとCSCC１．３×１０4個入れ、３７℃のＣＯ2インキュベータにて３日間細胞培養を行った。
培養後のセルの培養液を除去し、測定バッファー５００μＬを加え、水晶振動子装置にセットし、２７ＭＨｚで発振させて振動数を測定した。その測定結果を図４に示す。
図４から、コラーゲンを固定化した状態の周波数（同図中のＡ）は一定であり、細胞を培養した後の振動数は、低下していることがわかる（同図中のＢ）。
Example 2: Immobilization on a piezoelectric element electrode using a collagen adhesive layer of CSCC and measurement of volume change A Piranha solution (30% hydrogen peroxide solution: concentrated sulfuric acid = 1) is applied to the electrode of the piezoelectric element of the biosensor cell. : 3) was applied in an appropriate amount, allowed to stand for 5 to 10 minutes, and further the same operation was performed to clean the electrode part. Next, a collagen solution (Cellmatrix Type I-C manufactured by Nitta Gelatin Co., Ltd.) was diluted 10 times, thinly applied on the electrode of the piezoelectric element, dried, and then sterilized by irradiation with ultraviolet rays for about 10 minutes.
The cell was washed with a culture solution, and 500 μL of a measurement buffer (Tris / MOPS: 10 mM, NaCl: 135 mM, KCl: 5 mM, glucose: 5 mM) was added. Then, the cell was set in a crystal resonator device (Affinix Q4: manufactured by Initium Co., Ltd.), oscillated at 27 MHz, and the frequency was measured. The result is shown in FIG.
The measurement buffer was removed from the cell, sprayed with 70% ethanol, and then irradiated with ultraviolet rays for about 10 minutes to sterilize. 320 μL of the culture solution and 1.3 × 10 4 CCCC were placed in a sterilized cell, and the cells were cultured in a CO 2 incubator at 37 ° C. for 3 days.
The culture solution in the cell after the culture was removed, 500 μL of a measurement buffer was added, the cell was set in a crystal resonator device, and the frequency was measured by oscillating at 27 MHz. The measurement results are shown in FIG.
FIG. 4 shows that the frequency of immobilized collagen (A in the figure) is constant, and the frequency after the cells are cultured is reduced (B in the figure).
１ 細胞容積の測定装置
２ 発振装置
３ ネットワークアナライザ
４ コンピュータ
５ ケーブル
６ ケーブル
７ 水晶振動子
８ 円形状の結晶板
９ 第一の金電極（表面）
１０ 第二の金電極（裏面）
１１ 樹脂カバー
１２ 電極上に培養された細胞
１３ 信号供給回路
１４ 測定回路
１５ セル
１６ 細胞等張液
DESCRIPTION OF SYMBOLS 1 Cell volume measuring apparatus 2 Oscillator 3 Network analyzer 4 Computer 5 Cable 6 Cable 7 Crystal oscillator 8 Circular crystal plate 9 First gold electrode (surface)
10 Second gold electrode (back)
11 resin cover 12 cell cultured on electrode 13 signal supply circuit 14 measurement circuit 15 cell 16 cell isotonic solution
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