JP4742973B2 - Cell electrophysiology measuring device and method of manufacturing the same - Google Patents

Cell electrophysiology measuring device and method of manufacturing the same Download PDF

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JP4742973B2
JP4742973B2 JP2006125516A JP2006125516A JP4742973B2 JP 4742973 B2 JP4742973 B2 JP 4742973B2 JP 2006125516 A JP2006125516 A JP 2006125516A JP 2006125516 A JP2006125516 A JP 2006125516A JP 4742973 B2 JP4742973 B2 JP 4742973B2
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hole
substrate
recess
measurement device
wall
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JP2007298351A5 (en
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章義 大島
将也 中谷
聡一郎 平岡
浩司 牛尾
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Panasonic Corp
Panasonic Holdings Corp
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Matsushita Electric Industrial Co Ltd
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Description

本発明は、薬剤に対する細胞の反応、或いは細胞が発する電気生理現象によって生じる電気化学的変化を測定するために用いられる細胞電気生理測定デバイスと、この細胞電気生理測定デバイスの製造方法に関する。   The present invention relates to a cell electrophysiology measuring device used for measuring an electrochemical change caused by a reaction of a cell to a drug or an electrophysiological phenomenon generated by the cell, and a method for producing the cell electrophysiology measuring device.

図１３に示すように、従来開示されている細胞電気生理測定デバイス１は、基板２と、この基板２の上部に設けた第一の貯留槽３と、基板２の下部に設けた第二の貯留槽４と、第一の貯留槽３および前記第二の貯留槽４にそれぞれ接続された第一の測定電極５および第二の測定電極６とを備えている。そして基板２は、上面に設けた凹部７と、この凹部７から基板２の下面まで繋がる貫通孔８とを有している。   As shown in FIG. 13, a cell electrophysiology measurement device 1 disclosed in the past includes a substrate 2, a first storage tank 3 provided on the upper portion of the substrate 2, and a second reservoir provided on the lower portion of the substrate 2. A storage tank 4, a first measurement electrode 5 and a second measurement electrode 6 connected to the first storage tank 3 and the second storage tank 4, respectively, are provided. The substrate 2 has a recess 7 provided on the upper surface, and a through hole 8 connected from the recess 7 to the lower surface of the substrate 2.

そしてこの細胞電気生理測定デバイス１は、第一の貯留槽３に被験体細胞９を包含する培養液１０ａを注入し、貫通孔８の上方から加圧、あるいは下方から減圧することで培養液１０ａを吸引し、被験体細胞９を凹部７に捕捉することができる。そして例えばこの被験体細胞９の上から薬剤を投与し、第一の貯留槽３の培養液１０ａと第二の貯留槽４の培養液１０ｂとの電位差を第一の測定電極５および第２の測定電極６で測定し、検出装置１１を用いて比較・分析することで被験体細胞９の薬理反応を判断することができる。   And this cell electrophysiology measuring device 1 inject | pours into the 1st storage tank 3 the culture solution 10a which contains a test subject cell 9, and pressurizes from the upper direction of the through-hole 8, or depressurizes from the downward direction, The culture solution 10a The subject cell 9 can be captured in the recess 7. Then, for example, a drug is administered from above the subject cell 9, and the potential difference between the culture solution 10 a in the first storage tank 3 and the culture solution 10 b in the second storage tank 4 is determined as the first measurement electrode 5 and the second measurement electrode 5. The pharmacological reaction of the subject cell 9 can be determined by measuring with the measurement electrode 6 and comparing and analyzing using the detection device 11.

なお、この出願の発明に関する先行技術文献情報としては、特許文献１や非特許文献１などが知られている。
特開２００４−６９３０９号公報 Ｔ.Ｌehnertetal, Ｍicro Ｔotal Ａnalysis Ｓystems 2002，pp.28-30（２００４） As prior art document information relating to the invention of this application, Patent Document 1, Non-Patent Document 1, and the like are known.
JP 2004-69309 A T. Lehnertetal, Micro Total Analysis Systems 2002, pp. 28-30 (2004)

上記従来の構成では、細胞電気生理測定デバイス１の測定精度が低いという問題があった。   The conventional configuration has a problem that the measurement accuracy of the cell electrophysiological measurement device 1 is low.

これは被験体細胞９と貫通孔８の開口部８ａとの密着性が低く、被験体細胞９を境とした第一の貯留槽３と第二の貯留槽４との間の電気的絶縁性が十分に確保されていない為であった。そしてその結果、測定精度が低下するのであった。   This is because the adhesion between the subject cell 9 and the opening 8a of the through hole 8 is low, and the electrical insulation between the first reservoir 3 and the second reservoir 4 with the subject cell 9 as a boundary. This is because there is not enough. As a result, the measurement accuracy is lowered.

そこで本発明は、上記課題を解決し、細胞電気生理測定デバイス１における測定精度を高めることを目的とする。   SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to solve the above problems and increase the measurement accuracy in the cell electrophysiology measurement device 1.

この目的を達成するために本発明は、被験体細胞と密着させる貫通孔の開口部には、凹部の内部に向けて上方に突出した突出部を備え、この突出部は、その先端から湾曲して外方へ下降する凹部の内壁と、貫通孔の内壁とで形成されるものとした。   In order to achieve this object, in the present invention, the opening of the through-hole to be in close contact with the subject cell is provided with a protrusion protruding upward toward the inside of the recess, and the protrusion is curved from the tip. The inner wall of the concave part descending outward and the inner wall of the through hole are formed.

この構成により本発明は、被験体細胞と貫通孔開口部との密着性を高めることができる。   With this configuration, the present invention can improve the adhesion between the subject cell and the through-hole opening.

それは、前述の突出部によって被験体細胞と貫通孔開口部との接触面積が増大し、かつこの突出部が被験体細胞へ食い込み、接触抵抗が大きくなるためである。そしてその結果、細胞電気生理測定デバイスの測定精度を向上させることが出来るのである。   This is because the contact area between the subject cell and the through-hole opening is increased by the above-described protrusion, and the protrusion bites into the subject cell, thereby increasing the contact resistance. As a result, the measurement accuracy of the cell electrophysiology measurement device can be improved.

（実施の形態１）
以下、本実施の形態における細胞電気生理測定デバイスの構成を説明する。 (Embodiment 1)
Hereinafter, the configuration of the cell electrophysiology measurement device according to the present embodiment will be described.

図１の断面図に示すように、本実施の形態の細胞電気生理測定デバイス１２は、基板１３と、この基板１３の上部に設けられた、第一の貯留槽１４を有するウェル１５と、基板１３の下部に設けられた、第二の貯留槽１６を有するプレート１７と、第一の貯留槽１４内に設けられた第一の測定電極１８と、第二の貯留槽１６内であって、基板１３の下面に設けられた第二の測定電極１９と、これらの第一の測定電極１８および第二の測定電極１９により得られた電位を比較検出する検出装置２０とを備えている。   As shown in the cross-sectional view of FIG. 1, the cell electrophysiological measurement device 12 of the present embodiment includes a substrate 13, a well 15 having a first storage tank 14 provided on the substrate 13, and a substrate. 13, a plate 17 having a second storage tank 16, a first measurement electrode 18 provided in the first storage tank 14, and a second storage tank 16, A second measurement electrode 19 provided on the lower surface of the substrate 13 and a detection device 20 for comparing and detecting potentials obtained by the first measurement electrode 18 and the second measurement electrode 19 are provided.

なお、第一の貯留槽１４は培養液２１ａを貯留するためのものであり、第二の貯留槽１６は培養液２１ｂを貯留するためのものである。また第一の測定電極１８と第二の測定電極１９とは、培養液２１ａと培養液２１ｂとの電位差を検出するためのものである。   In addition, the 1st storage tank 14 is for storing the culture solution 21a, and the 2nd storage tank 16 is for storing the culture solution 21b. The first measurement electrode 18 and the second measurement electrode 19 are for detecting a potential difference between the culture solution 21a and the culture solution 21b.

そして基板１３は、その上面に、被験体細胞２２を捕捉保持する凹部２３と、この凹部２３の底部から基板１３下面までをつなぐ貫通孔２４とを有し、この貫通孔２４の開口部２４ａは、凹部２３の内部に向けて上方に突出した突出部２４ｂを有している。そしてこの突出部２４ｂは、その先端から湾曲して斜め外方へ下降する凹部２３の内壁と、この凹部２３の内壁と連続する貫通孔２４の内壁とで形成されている。本実施の形態では、図２に示すように、貫通孔２４は基板１３に対し垂直な円柱構造に形成し、突出部２４ｂは、その先端が鋭利なものではなく、上方に向かって丸みを帯びた湾曲面で形成されたものとした。また、突出部２４ｂの先端を、基板１３上面より下方に形成し、凹部２３に適切に被験体細胞（図１の２２）を捕捉できるようにしている。   And the board | substrate 13 has the recessed part 23 which capture | acquires and hold | maintains the subject cell 22 on the upper surface, and the through-hole 24 which connects from the bottom part of this recessed part 23 to the lower surface of the board | substrate 13, The opening part 24a of this through-hole 24 is , And has a protruding portion 24 b protruding upward toward the inside of the recess 23. The protrusion 24b is formed by an inner wall of the recess 23 that is curved from the tip and descends obliquely outward, and an inner wall of the through hole 24 that is continuous with the inner wall of the recess 23. In the present embodiment, as shown in FIG. 2, the through hole 24 is formed in a columnar structure perpendicular to the substrate 13, and the protrusion 24b is not sharp at the tip but rounds upward. It was formed with a curved surface. Further, the tip of the protrusion 24b is formed below the upper surface of the substrate 13 so that the subject cell (22 in FIG. 1) can be appropriately captured in the recess 23.

なお、図３の基板１３の斜視図に示すように、本実施の形態における細胞電気生理測定デバイス１２は、基板１３に前述の凹部２３および貫通孔２４を複数個形成している。また、基板１３としては単結晶シリコンを用い、この基板１３の表面にはシリコン酸化物からなる絶縁膜（図１１の３０）を形成している。   Note that, as shown in the perspective view of the substrate 13 in FIG. 3, the cell electrophysiological measurement device 12 in the present embodiment has a plurality of the aforementioned recesses 23 and through holes 24 formed in the substrate 13. Further, single crystal silicon is used as the substrate 13, and an insulating film (30 in FIG. 11) made of silicon oxide is formed on the surface of the substrate 13.

さらに、被験体細胞（図１の２２）としては哺乳類細胞を用いた。この場合、代表的な培養液２１ａとしては、Ｋ+イオンが１５５ｍＭ、Ｎａ+イオンが１２ｍＭ程度、Ｃｌ-イオンが４．２ｍＭ程度添加された電解液、培養液２１ｂとしてはＫ+イオンが４ｍＭ程度、Ｎａ+イオンが１４５ｍＭ程度、Ｃｌ-イオンが１２３ｍＭ程度添加された電解液が挙げられる。 Furthermore, mammalian cells were used as the subject cells (22 in FIG. 1). In this case, typical culture 21a, K + ions 155 mM, Na + ions of about 12 mM, Cl - ions electrolytic solution added about 4.2 mM, 4 mM approximately the K + ions as the culture solution 21b An electrolyte containing about 145 mM Na + ions and about 123 mM Cl − ions can be used.

次に、本発明の細胞電気生理測定デバイス１２の動作について説明する。   Next, the operation of the cell electrophysiology measuring device 12 of the present invention will be described.

図１に示すように、まず、第一の貯留槽１４を、被験体細胞２２を包含する第一の培養液２１ａで満たし、第一の貯留槽１４側から加圧（あるいは第二の貯留槽１６側から減圧）して第一の培養液２１ａを貫通孔２４の第二の貯留槽１６側へ流動させる。   As shown in FIG. 1, first, the first storage tank 14 is filled with a first culture solution 21a containing a subject cell 22, and pressurized (or a second storage tank) from the first storage tank 14 side. The first culture solution 21a is caused to flow to the second reservoir 16 side of the through hole 24 by reducing the pressure from the 16th side.

するとこの第一の培養液２１ａの流動に伴い、被験体細胞２２が凹部２３に捕捉される。この捕捉された被験体細胞２２によって、第一の培養液２１ａと第二の培養液２１ｂとが電気的に分離される。   Then, the subject cells 22 are captured in the recesses 23 with the flow of the first culture solution 21a. By the captured subject cell 22, the first culture solution 21a and the second culture solution 21b are electrically separated.

次に第二の貯留槽１６側から吸引、もしくは薬剤（例えばナイスタチンなど）を投入することで被験体細胞２２の第二の貯留槽１６側の細胞膜に微細小孔を形成する。   Next, a fine pore is formed in the cell membrane of the subject cell 22 on the second reservoir 16 side by suction or injection of a drug (for example, nystatin) from the second reservoir 16 side.

その後第一の貯留槽１４側から被験体細胞２２に対して刺激となりうる行為を施す。この刺激の種類としては、例えば化学薬品、毒薬などの化学的刺激に加え、機械的変位、光、熱、電気、電磁波などの物理的刺激が挙げられる。   Thereafter, an action that can be a stimulus to the subject cell 22 is performed from the first reservoir 14 side. Examples of the type of stimulation include physical stimulation such as mechanical displacement, light, heat, electricity, and electromagnetic waves in addition to chemical stimulation such as chemicals and poisons.

ここで被験体細胞２２がこれらの刺激に対して反応を示せば被験体細胞２２の細胞膜内に存在するイオンチャネルが活性または拮抗し、このイオンチャネルを介してイオンの移動量が変化するため、この被験体細胞２２を通過した第二の培養液２１ｂの電位に変化が生じることになる。   Here, if the subject cell 22 shows a response to these stimuli, the ion channel existing in the cell membrane of the subject cell 22 is activated or antagonized, and the amount of ion movement changes through this ion channel. A change occurs in the potential of the second culture solution 21b that has passed through the subject cell 22.

この電位を第一の測定電極１８と第二の測定電極１９で検出し、検出装置２０で比較・分析することで、薬剤が被験体細胞２２に対して有効性を有するかどうかなどを判断するのである。   This potential is detected by the first measurement electrode 18 and the second measurement electrode 19 and compared / analyzed by the detection device 20 to determine whether or not the drug has an effect on the subject cell 22. It is.

このとき、被験体細胞２２が高い密着性を持って貫通孔２４の開口部２４ａおよび凹部２３に接触保持されることで低いバックグラウンドノイズで被験体細胞２２の電気生理現象を測定することが可能となる。   At this time, the electrophysiological phenomenon of the subject cell 22 can be measured with low background noise by holding the subject cell 22 in contact with the opening 24a and the recess 23 of the through hole 24 with high adhesion. It becomes.

次に、本実施の形態における細胞電気生理測定デバイス１２の製造方法を説明する。   Next, the manufacturing method of the cell electrophysiology measuring device 12 in this Embodiment is demonstrated.

まず図４に示すように、基板１３の上面にフォトリソグラフィーによりレジストマスク２６を形成する。このレジストマスク２６には、所望の貫通孔２４の開口部２４ａと略同形状のマスクホール２７を形成しておく。   First, as shown in FIG. 4, a resist mask 26 is formed on the upper surface of the substrate 13 by photolithography. A mask hole 27 having substantially the same shape as the opening 24 a of the desired through hole 24 is formed in the resist mask 26.

次に図５に示すように基板１３をドライエッチングして貫通孔２４を形成する。この時のエッチングガスとしては、エッチングを促進するガス（以下エッチングガスという）とエッチングを抑制するガスと（以下抑制ガスという）とを交互に用いる。   Next, as shown in FIG. 5, the substrate 13 is dry-etched to form a through hole 24. As the etching gas at this time, a gas for promoting etching (hereinafter referred to as an etching gas), a gas for suppressing etching (hereinafter referred to as a suppressing gas) are alternately used.

エッチングガスとしてはＳＦ6、ＣＦ4、ＸｅＦ2などがある。また、抑制ガスとしてはＣＨＦ3、Ｃ4Ｆ8などが挙げられる。 Examples of the etching gas include SF 6 , CF 4 , and XeF 2 . In addition, examples of the suppression gas include CHF 3 and C 4 F 8 .

これらのエッチングガスにより基板１３を少しエッチングし、その後抑制ガスによりシリコン面に保護膜を形成するステップを繰り返すことで、基板１３の上面に対して垂直方向のみのエッチングが可能となる。   Etching only in a direction perpendicular to the upper surface of the substrate 13 is possible by repeating the steps of slightly etching the substrate 13 with these etching gases and then forming a protective film on the silicon surface with the suppression gas.

そして図６に示すように、貫通孔２４を形成後、抑制ガスを吹き付け、貫通孔２４側壁にポリマーからなる保護膜２５を形成し、レジストマスク２６を除去する。なお、この保護膜２５を形成するその他の方法として、貫通孔２４を形成し、レジストマスク２６を除去した後に熱酸化法、ＣＶＤ法、スパッタリング法等の成膜方法を用いて酸化物からなる保護膜２５を形成する方法がある。この後者の方法では、後述する凹部２３を形成する工程で利用するレジストマスク（図７の２８）のエッチングホール（図７の２９）に沿って基板１３の上面に成膜された保護膜２５をエッチングする必要がある。この際、エッチングには上面のみのエッチングが可能なドライエッチングが有効となる。   Then, as shown in FIG. 6, after forming the through hole 24, a suppression gas is sprayed to form a protective film 25 made of a polymer on the side wall of the through hole 24, and the resist mask 26 is removed. As another method for forming this protective film 25, after forming the through hole 24 and removing the resist mask 26, a protective film made of oxide using a film forming method such as a thermal oxidation method, a CVD method, or a sputtering method is used. There is a method of forming the film 25. In this latter method, the protective film 25 formed on the upper surface of the substrate 13 is formed along the etching hole (29 in FIG. 7) of the resist mask (28 in FIG. 7) used in the step of forming the recess 23 described later. It needs to be etched. At this time, dry etching capable of etching only the upper surface is effective for the etching.

なおこの保護膜２５によって、後の凹部２３形成の際のドライエッチングの影響を受けず、貫通孔２４の形状を保つことができる。   The protective film 25 allows the shape of the through hole 24 to be maintained without being affected by dry etching when the recess 23 is formed later.

次に図７に示すように、基板１３に貫通孔２４の開口部２４ａの外郭に環状のマスクホール２９を有する新たなレジストマスク２８を形成し、ＳＦ6またはＣＦ4またはＸｅＦ2またはＣｌＦ3またはＮＦ3またはこれらの混合ガスのいずれかを用いたエッチングガスでＳｉのドライエッチングを施す。 Next, as shown in FIG. 7, a new resist mask 28 having an annular mask hole 29 is formed on the substrate 13 around the opening 24a of the through hole 24, and SF 6 or CF 4 or XeF 2 or ClF 3 or Si dry etching is performed with an etching gas using either NF 3 or a mixed gas thereof.

すると図８に示すように、環状のマスクホール２９の部分を中心に等方的にエッチングが進み、凹部２３が形成される。   Then, as shown in FIG. 8, the etching proceeds isotropically around the annular mask hole 29, and the recess 23 is formed.

このとき、図９に示すように、貫通孔２４の内壁と凹部２３の内壁とが連結し、突出部２４ｂの先端が基板１３の上面より下方になるまでエッチングを施した後、レジストマスク２８を除去する。この時同時に保護膜２５も除去され、図１０に示すように、貫通孔２４の開口部２４ａには凹部２３内方に向かって上方に突出した突出部２４ｂを形成することが出来る。   At this time, as shown in FIG. 9, after etching is performed until the inner wall of the through hole 24 and the inner wall of the recess 23 are connected and the tip of the protruding portion 24b is below the upper surface of the substrate 13, the resist mask 28 is removed. Remove. At the same time, the protective film 25 is also removed, and as shown in FIG. 10, a protruding portion 24 b protruding upward toward the inside of the recess 23 can be formed in the opening 24 a of the through hole 24.

さらにその後、図１１に示すように、熱酸化法、ＣＶＤ法、スパッタリング法等の成膜方法を用いることで構造体表面にシリコン酸化物からなる絶縁膜３０を形成する。この絶縁膜３０は、その他シリコン窒化物膜などによって形成してもよい。この絶縁膜３０が成膜されることで突出部２４ｂの先端は丸みを帯びた湾曲面で形成される。   Further, as shown in FIG. 11, an insulating film 30 made of silicon oxide is formed on the structure surface by using a film forming method such as a thermal oxidation method, a CVD method, or a sputtering method. The insulating film 30 may be formed of other silicon nitride film or the like. By forming the insulating film 30, the tip of the protruding portion 24b is formed with a rounded curved surface.

その他突出部２４ｂの先端を上記のような湾曲面で形成するには、ドライエッチングあるいはウェットエッチング加工や、不活性ガスによるアニール処理で鋭利なエッジ部分を丸くする方法がある。   In addition, in order to form the tip of the protruding portion 24b with the curved surface as described above, there is a method of rounding a sharp edge portion by dry etching or wet etching or annealing with an inert gas.

本実施の形態における効果を以下に説明する。   The effect in this Embodiment is demonstrated below.

まず、本実施の形態では、細胞電気生理測定デバイス１２の測定精度を高めることが出来る。   First, in the present embodiment, the measurement accuracy of the cell electrophysiological measurement device 12 can be increased.

これは、図１に示すように、貫通孔２４の開口部２４ａには、凹部２３の内方に向けて上方に突出した突出部２４ｂがあり、この突出部２４ｂは、その先端から湾曲して外方へ下降する凹部２３の内壁と、貫通孔２４の内壁とで形成されていることに由来する。   As shown in FIG. 1, the opening 24a of the through hole 24 has a protrusion 24b that protrudes upward toward the inside of the recess 23. The protrusion 24b is curved from the tip thereof. This is derived from the fact that the inner wall of the recess 23 descending outward and the inner wall of the through hole 24 are formed.

すなわち、突出部２４ｂは上記構成によってなだらかに湾曲する傾斜面で凹部２３と一体となっていることから、被験体細胞２２は、この凹部２３内壁の湾曲面に沿って、隙間が極力ないよう密着することができ、被験体細胞２２と貫通孔２４の開口部２４ａとの当接面積を増すことができるためである。   That is, since the protrusion 24b is an inclined surface that is gently curved with the above configuration and is integrated with the recess 23, the subject cell 22 adheres so that there is as little gap as possible along the curved surface of the inner wall of the recess 23. This is because the contact area between the subject cell 22 and the opening 24a of the through hole 24 can be increased.

さらにこの突出部２４ｂが被験体細胞２２を吸引する方向と逆向きに形成されていることから、吸引の際に接触抵抗が増し、突出部２４ｂが被験体細胞２２に食い込むような形となる。   Further, since the protrusion 24b is formed in the direction opposite to the direction in which the subject cell 22 is sucked, the contact resistance increases during the suction, and the protrusion 24b bites into the subject cell 22.

そしてこのような当接面積の増大と食い込みにより、貫通孔２４開口部と被験体細胞２２との密着性が増し、この結果測定精度を向上させることが出来るのである。   And by such an increase in contact area and biting in, the adhesion between the opening of the through hole 24 and the subject cell 22 increases, and as a result, the measurement accuracy can be improved.

また、本実施の形態では、図２に示すように突出部２４ｂの先端を、上方に向けて丸みを帯びた湾曲面で構成したことから、被験体細胞２２が突出部２４ｂに食い込んだ際も、被験体細胞２２の細胞膜の破損を抑制することができる。   Further, in the present embodiment, as shown in FIG. 2, the tip of the protruding portion 24b is configured with a curved surface that is rounded upward, so that the subject cell 22 bites into the protruding portion 24b. The damage of the cell membrane of the subject cell 22 can be suppressed.

さらに図１１に示すように、凹部２３および貫通孔２４の内壁を含む基板１３表面全体をシリコン酸化物からなる絶縁膜３０で被覆したことにより、微細加工に適したシリコン基板１３を用いつつ、基板１３上面と下面との間の絶縁性を確保することができる。そしてこのシリコン酸化物膜は同時に親水性を有することから、貫通孔２４の開口部２４ａ近傍において被験体細胞２２の密着性をさらに向上させることができる。   Further, as shown in FIG. 11, the entire surface of the substrate 13 including the inner walls of the recesses 23 and the through holes 24 is covered with an insulating film 30 made of silicon oxide, so that the substrate can be used while using the silicon substrate 13 suitable for fine processing. 13 Insulation between the upper surface and the lower surface can be ensured. Since this silicon oxide film is hydrophilic at the same time, the adhesion of the subject cell 22 can be further improved in the vicinity of the opening 24a of the through hole 24.

また、貫通孔２４内壁も親水性の高いシリコン酸化物膜で被覆することで貫通孔２４内部における流路抵抗が低減され、吸引ポンプなどによる吸引効率が向上し、被験体細胞２２が貫通孔２４の開口部２４ａ近傍および凹部２３に密着し易くなる。   Further, the inner wall of the through hole 24 is also covered with a highly hydrophilic silicon oxide film, thereby reducing the flow resistance inside the through hole 24, improving the suction efficiency by a suction pump or the like, and allowing the subject cell 22 to pass through the through hole 24. It becomes easy to closely adhere to the vicinity of the opening 24a and the recess 23.

なお、本実施の形態では絶縁膜３０としてシリコン酸化物を用いたが、他の酸化膜、窒化膜など親水性を有する絶縁膜３０を用いれば上記と同様の効果を得られる。   Although silicon oxide is used as the insulating film 30 in this embodiment, the same effect as described above can be obtained by using a hydrophilic insulating film 30 such as another oxide film or nitride film.

（実施の形態２）
本実施の形態と実施の形態１との違いは、図１２に示すように貫通孔３１の形状を、貫通孔３１の開口部３１ａから基板１３下面に向けて広がったテーパ構造にしたことである。 (Embodiment 2)
The difference between the present embodiment and the first embodiment is that the shape of the through hole 31 is a tapered structure that spreads from the opening 31a of the through hole 31 toward the lower surface of the substrate 13 as shown in FIG. .

これにより貫通孔３１の開口部３１ａから基板１３下面に行くにつれて培養液２１ｂの流束密度が徐々に減少するため、安定して培養液２１ｂを吸引することができる。そしてその結果、被験体細胞２２を貫通孔３１の開口部３１ａに的確に吸引することができる。   As a result, the flux density of the culture solution 21b gradually decreases from the opening 31a of the through-hole 31 toward the lower surface of the substrate 13, so that the culture solution 21b can be stably sucked. As a result, the subject cell 22 can be accurately sucked into the opening 31 a of the through hole 31.

なお、本実施の形態における貫通孔３１は、この貫通孔３１を形成するドライエッチング工程において、エッチングを促進するガス（エッチングガス）とエッチングを抑制するガス（抑制ガス）の割合を、エッチングが進むに従って（貫通孔３１の下部に行くに従って）、エッチングガスの割合を増やせばよい。   In the through hole 31 in the present embodiment, in the dry etching process for forming the through hole 31, etching proceeds at a ratio of a gas that promotes etching (etching gas) and a gas that suppresses etching (suppressing gas). According to (according to the lower part of the through hole 31), the ratio of the etching gas may be increased.

本発明の細胞電気生理測定デバイスは、細胞の薬剤に対する反応、或いは細胞が発する電気生理現象によって生じる電気化学的変化を、高精度で測定するのに有用である。   The cell electrophysiology measuring device of the present invention is useful for measuring with high accuracy an electrochemical change caused by a reaction of a cell to a drug or an electrophysiological phenomenon generated by the cell.

本発明の細胞電気生理測定デバイスの断面図Sectional view of the cell electrophysiology measuring device of the present invention 本発明の細胞電気生理測定デバイスの要部拡大模式断面図The principal part expansion schematic cross section of the cell electrophysiology measuring device of this invention 本発明の細胞電気生理測定デバイスの斜視図The perspective view of the cell electrophysiology measuring device of this invention 本発明の細胞電気生理測定デバイスの製造工程を示す断面図Sectional drawing which shows the manufacturing process of the cell electrophysiology measuring device of this invention 同断面図Sectional view 同断面図Sectional view 同断面図Sectional view 同断面図Sectional view 同断面図Sectional view 同断面図Sectional view 同断面図Sectional view 本発明の細胞電気生理測定デバイスの要部断面図Sectional drawing of the principal part of the cell electrophysiology measuring device of the present invention 従来の細胞電気生理測定デバイスの断面図Sectional view of a conventional cell electrophysiology measurement device

符号の説明Explanation of symbols

１ 細胞電気生理測定デバイス
２ 基板
３ 第一の貯留槽
４ 第二の貯留槽
５ 第一の測定電極
６ 第二の測定電極
７ 凹部
８ 貫通孔
８ａ 開口部
９ 被験体細胞
１０ａ 培養液
１０ｂ 培養液
１１ 検出装置
１２ 細胞電気生理測定デバイス
１３ 基板
１４ 第一の貯留槽
１５ ウェル
１６ 第二の貯留槽
１７ プレート
１８ 第一の測定電極
１９ 第二の測定電極
２０ 検出装置
２１ａ 第一の培養液
２１ｂ 第二の培養液
２２ 被験体細胞
２３ 凹部
２４ 貫通孔
２４ａ 開口部
２４ｂ 突出部
２５ 保護膜
２６ レジストマスク
２７ マスクホール
２８ レジストマスク
２９ マスクホール
３０ 絶縁膜
３１ 貫通孔
３１ａ 開口部 DESCRIPTION OF SYMBOLS 1 Cell electrophysiology measuring device 2 Board | substrate 3 1st storage tank 4 2nd storage tank 5 1st measurement electrode 6 2nd measurement electrode 7 Recessed part 8 Through-hole 8a Opening part 9 Subject cell 10a Culture solution 10b Culture solution DESCRIPTION OF SYMBOLS 11 Detection apparatus 12 Cell electrophysiology measurement device 13 Substrate 14 1st storage tank 15 Well 16 2nd storage tank 17 Plate 18 1st measurement electrode 19 2nd measurement electrode 20 Detection apparatus 21a 1st culture solution 21b 1st Second culture solution 22 Subject cell 23 Recessed portion 24 Through hole 24a Opening portion 24b Protruding portion 25 Protective film 26 Resist mask 27 Mask hole 28 Resist mask 29 Mask hole 30 Insulating film 31 Through hole 31a Opening portion

Claims (7)

基板と、
この基板の上部に設けた第一の貯留槽と、
前記基板の下部に設けた第二の貯留槽と、
前記第一の貯留槽および前記第二の貯留槽との電位差を検出するためそれぞれに設けられた測定電極とを備え、
前記基板は、
この基板の上面に設けた凹部と、
この凹部の底部から開口し前記基板の下面まで繋がる貫通孔とを有し、
この貫通孔の開口部は、
前記凹部の内部に向けて上方に突出した突出部を備え、
この突出部は、
その先端から湾曲して外方へ下降する前記凹部の内壁と、
前記貫通孔の内壁とで形成され、
前記突出部の先端は、前記基板上面より下方にある細胞電気生理測定デバイス。 A substrate,
A first storage tank provided on top of the substrate;
A second storage tank provided at the bottom of the substrate;
A measuring electrode provided for each to detect a potential difference between the first storage tank and the second storage tank;
The substrate is
A recess provided on the upper surface of the substrate;
Having a through hole that opens from the bottom of the recess and leads to the lower surface of the substrate,
The opening of this through hole is
Protruding portion protruding upward toward the inside of the recess,
This protrusion is
An inner wall of the recess curved from the tip and descending outward;
Formed with the inner wall of the through hole ,
The tip of the protrusion,胞電vapor physiological measurement device thin in from below the substrate top surface. 前記突出部の先端は、
上方に丸みを帯びた湾曲面で形成されている請求項１に記載の細胞電気生理測定デバイス。 The tip of the protrusion is
The cell electrophysiological measurement device according to claim 1, wherein the cell electrophysiological measurement device is formed with an upwardly rounded curved surface. 前記貫通孔は、
前記基板に対して垂直な円柱構造、
または前記開口部から前記基板の下面に向けて口径が広がるテーパ構造である請求項１または２に記載の細胞電気生理測定デバイス。 The through hole is
A cylindrical structure perpendicular to the substrate,
Or cellular electrophysiological measurement device of the serial placement through the opening in claim 1 or 2 which is tapered structure diameter widens toward the lower surface of the substrate. 前記基板はシリコンからなり、
この基板表面は絶縁膜で被覆されている請求項１から３のいずれか一つに記載の細胞電気生理測定デバイス。 The substrate is made of silicon;
The cell electrophysiological measurement device according to any one of claims 1 to 3 , wherein the substrate surface is coated with an insulating film. 基板の上面から下面に向けてドライエッチングで貫通孔を形成し、
次にこの貫通孔の内壁に保護膜を形成し、
その後前記基板上において、前記貫通孔の外郭に環状のエッチングホールを有するマスクを形成し、
このマスク上方からドライエッチングで凹部を形成する工程からなり、
前記凹部を形成するドライエッチング工程は、
前記凹部の内壁と前記貫通孔の内壁とが繋がるようにドライエッチングし、前記貫通孔の開口部に前記凹部の内部に向けて上方に突出する突出部を形成する細胞電気生理測定デバイスの製造方法。 A through hole is formed by dry etching from the upper surface to the lower surface of the substrate,
Next, a protective film is formed on the inner wall of the through hole,
Thereafter, on the substrate, a mask having an annular etching hole is formed around the through-hole,
It consists of a step of forming a recess by dry etching from above the mask,
The dry etching process for forming the concave portion includes:
A method for manufacturing a cell electrophysiological measurement device, wherein dry etching is performed so that the inner wall of the recess and the inner wall of the through-hole are connected, and a protrusion protruding upward toward the inside of the recess is formed at the opening of the through-hole. . 前記貫通孔内壁に保護膜を形成する工程において、
前記貫通孔の内壁にはポリマー膜またはＳｉＯ2のいずれかを形成する請求項５に記載の細胞電気生理測定デバイスの製造方法。 In the step of forming a protective film on the inner wall of the through hole,
Method for manufacturing a cellular electrophysiological measurement device of claim 5 on an inner wall of the through hole to form either a polymeric film or SiO 2. 前記凹部を形成するドライエッチング工程において、
エッチングガスにＣＦ4またはＳＦ6またはＸｅＦ2またはＣｌＦ3またはＮＦ3またはこれらの混合ガスのいずれかを用いる請求項５または６に記載の細胞電気生理測定デバイスの製造方法。 In the dry etching process for forming the recess,
The method for producing a cell electrophysiological measurement device according to claim 5 or 6 , wherein any one of CF 4, SF 6, XeF 2, ClF 3, NF 3, or a mixed gas thereof is used as an etching gas.
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