JP2011152324A - Electrode for intracerebral substance measurement - Google Patents
Electrode for intracerebral substance measurement Download PDFInfo
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本発明は、哺乳類の脳内に刺入して脳内アミン等の脳内物質の変動等の電気化学的測定に有用な脳内物質測定用電極及びそれを用いた脳内物質変動の測定法に関する。 The present invention relates to an electrode for measuring a substance in the brain that is inserted into a mammalian brain and is useful for electrochemical measurement of a substance in the brain such as an amine in the brain, and a method for measuring the substance fluctuation in the brain using the same. About.
脳内アミン量は、各種中枢神経系疾患に深く関与しており、その変動をモニターすることは疾患の診断だけでなく、疾患や脳内アミンの機能を研究する上で極めて重要である。脳内アミンの測定法としては、脳組織ホモジネート除蛋白液のHPLC解析、マイクロダイアリシス法等があるが、これらの手段では秒単位で変動する被験体の行動に追随した高速では脳内アミン変動を測定できない。一方、微小電極を脳内に刺入又は留置して電気化学的に脳内アミンの変動を測定する手段は、オンタイムで脳内アミン濃度の変動が高速測定できる点で有用である。 The amount of amine in the brain is deeply involved in various central nervous system diseases, and monitoring its fluctuation is extremely important not only for diagnosing the disease but also for studying the function of the disease and the amine in the brain. Methods for measuring brain amines include HPLC analysis of brain tissue homogenate deproteinized solution, microdialysis method, etc., but these methods change the amine in the brain at high speed following the subject's behavior that varies in seconds. Cannot be measured. On the other hand, a means for electrochemically measuring changes in brain amine by inserting or placing a microelectrode in the brain is useful in that the change in amine concentration in the brain can be measured at high speed.
脳内アミン測定用の電極としては、脳内に刺入できるカーボンファイバー電極を用いてげっ歯の脳内ドパミンを測定する技術が報告されている（非特許文献１及び２）。しかし、カーボンファイバーは折れやすく、操作上の困難を伴う。また、電極が折れた場合、その断片が脳内に残存してしまい、摘出する方法は無く安全上好ましくない。これに対し、タングステン針の表面に１％ホウ素混入ダイヤモンドを蒸着した電気化学測定用電極が報告されている（非特許文献３）。 As an electrode for measuring an amine in the brain, a technique for measuring the intracerebral dopamine in a rodent using a carbon fiber electrode that can be inserted into the brain has been reported (Non-patent Documents 1 and 2). However, carbon fibers are easy to break and are difficult to operate. In addition, when the electrode is broken, the fragment remains in the brain, and there is no method for extraction, which is not preferable for safety. On the other hand, an electrode for electrochemical measurement in which 1% boron mixed diamond is deposited on the surface of a tungsten needle has been reported (Non-patent Document 3).
しかしながら、タングステン針の表面に１％ホウ素混入ダイヤモンドを蒸着した微小電極の感度は十分でなく、強い電気刺激により強制的に放出させた脳内アミンの測定には使用できても、生理的な条件での高感度測定が要求される場合に適用できるものではなかった。また、物理的強度とサイズにおいてマウスのような小実験動物に使用できても、サルやヒトのような大きな脳に適用できるものではなかった。
従って本発明の課題は、ヒトを含む大型動物の脳に刺入しても折れない十分な強度を有し、かつ高感度に脳内物質を測定できる微小電極を提供することにある。
However, the sensitivity of the microelectrode deposited with 1% boron-contained diamond on the surface of tungsten needle is not sufficient, and it can be used for the measurement of brain amines forcedly released by strong electrical stimulation. It was not applicable when high-sensitivity measurement was required. Moreover, even if it can be used for a small experimental animal such as a mouse in physical strength and size, it cannot be applied to a large brain such as a monkey or a human.
Accordingly, an object of the present invention is to provide a microelectrode having sufficient strength that cannot be broken even when inserted into the brain of a large animal including a human and capable of measuring a substance in the brain with high sensitivity.
そこで本発明者は、脳内で折れない十分な強度と高感度とを両立すべく種々検討した結果、高強度金属針の表面に高濃度ホウ素混入ダイヤモンドを蒸着し、電極として作用する部分を直径０．０１〜０．３ｍｍ、長さ０．０２〜２．０ｍｍの微小針状とし、それ以外の部分を絶縁部とすれば、大型動物の脳内に刺入しても折れ難く、かつ高感度で脳内物質の測定が可能であり、かつ操作性も良好であることを見出し、本発明を完成した。 Therefore, as a result of various studies to achieve both sufficient strength that does not break in the brain and high sensitivity, the present inventor deposited high-concentration boron-contaminated diamond on the surface of a high-strength metal needle, and the portion that acts as an electrode has a diameter. If it is made into a fine needle shape of 0.01 to 0.3 mm and a length of 0.02 to 2.0 mm and the other part is an insulating part, it is difficult to break even when inserted into the brain of a large animal, and high It was found that the substance in the brain can be measured with sensitivity and the operability is good, and the present invention has been completed.
すなわち、本発明は、高強度金属針の表面に３〜８％ホウ素原料混入ダイヤモンドが蒸着してなり、直径０．０１〜０．３ｍｍ、長さ０．０２〜２．０ｍｍの微小針状電極部を有することを特徴とする脳内物質測定用電極を提供するものである。
また、本発明は、上記電極を脳内に刺入し、定電位アンペロメトリー及びサイクリックボルタメトリーにより脳内物質の変動を測定することを特徴とする脳内物質変動の測定方法を提供するものである。
That is, the present invention is a fine needle electrode having a diameter of 0.01 to 0.3 mm and a length of 0.02 to 2.0 mm, in which 3 to 8% boron raw material mixed diamond is deposited on the surface of a high-strength metal needle. An electrode for measuring a substance in the brain is provided.
The present invention also provides a method for measuring substance fluctuations in the brain, wherein the electrode is inserted into the brain, and the substance fluctuations in the brain are measured by constant potential amperometry and cyclic voltammetry. Is.
本発明の電極は、微小でありながら、高い強度と高い感度を併せ持っており、ヒトを含む哺乳動物の脳内に刺入しても折れることなく、長時間の脳内物質測定を可能とする。従って、本発明の電極は、モノアミン系の障害により社会生活が困難な患者を支援するため、脳内モノアミン変動を日常的にモニターするための医療用電極となり得る。また、大型動物を用いた脳疾患を治療するための薬理学的、生理学的研究に用いるための研究用電極にもなり得る。 The electrode of the present invention has both high strength and high sensitivity even though it is minute, and enables long-term measurement of substances in the brain without breaking even when inserted into the brain of mammals including humans. . Therefore, the electrode of the present invention can be used as a medical electrode for daily monitoring of fluctuations in monoamines in the brain because it supports patients who have difficulty in social life due to monoamine-based disorders. It can also be a research electrode for use in pharmacological and physiological studies to treat brain diseases using large animals.
うつ病患者の脳内セロトニン変動を医師や家族が知ることができれば、自殺を未然に防ぐことができる。また、パニック障害の患者がモノアミンレベルの上昇を知り職場の同僚に知らせることができれば無理のない社会復帰の助けとなる。近年、脳神経系の治療において、患者脳の直接解剖学的観察を可能としたＭＲＩ診察技術の寄与は計り知れない。さらに、脳内に電極を留置し刺激する脳深部刺激（ＤＢＳ）治療法までも急速に普及している（非特許文献４）。従って、治療効果が明らかであれば、侵襲的脳手術でさえもパーキンソン病患者が喜んで受け入れるという実績が積み上げられつつあり、本発明の電極を用いた脳内記録電極の植え込みも現実的な治療法の一つとなりうる。 If doctors and family members can know serotonin changes in the brain of depressed patients, suicide can be prevented. Also, if a patient with panic disorder knows that monoamine levels have risen and can inform colleagues in the workplace, it will help them to reinstate themselves. In recent years, in the treatment of the cranial nervous system, the contribution of MRI examination technology that enables direct anatomical observation of the patient's brain has been immeasurable. Furthermore, a deep brain stimulation (DBS) treatment method in which an electrode is placed and stimulated in the brain is rapidly spreading (Non-patent Document 4). Therefore, if the therapeutic effect is clear, the track record that patients with Parkinson's disease are willing to accept even invasive brain surgery is being accumulated, and implantation of the recording electrode in the brain using the electrode of the present invention is also a realistic treatment It can be one of the laws.
本発明の電極の電極部は、高強度金属針の表面に３〜８％ホウ素原料混入ダイヤモンドが蒸着してなり、直径０．０１〜０．３ｍｍ、長さ０．０２〜２．０ｍｍの微小針状である。ここで高強度金属としては、タングステン、モリブデンが用いられるが、タングステンが強度、安全性、ダイヤモンド成長の基板となる等の点で特に好ましい。当該高強度金属針の長さは着脱により調節可能で、特に１０〜２０ｃｍ程度に延長可能とすることが電極マニピュレーター操作性の点で好ましい。 The electrode portion of the electrode of the present invention is formed by depositing 3-8% boron raw material mixed diamond on the surface of a high-strength metal needle, having a diameter of 0.01-0.3 mm and a length of 0.02-2.0 mm. It is acicular. Here, tungsten and molybdenum are used as the high-strength metal, and tungsten is particularly preferable in terms of strength, safety, and a diamond growth substrate. The length of the high-strength metal needle can be adjusted by attaching and detaching, and in particular, it is preferable from the viewpoint of electrode manipulator operability that it can be extended to about 10 to 20 cm.
本発明の電極の電極部は、上記１０ｃｍ以上の高強度金属針の先端部長さ０．０２〜２．０ｍｍ部分であるのが好ましく、さらに先端部長さ０．２〜２．０ｍｍ部分、特に先端部長さが０．３〜０．７ｍｍ部分であるのが好ましい。なお、高強度金属針の電極部以外の部分は、樹脂により絶縁されているのが好ましい。 The electrode portion of the electrode of the present invention preferably has a tip portion length of 0.02 to 2.0 mm, and more preferably a tip portion length of 0.2 to 2.0 mm, particularly the tip. The part length is preferably 0.3 to 0.7 mm. In addition, it is preferable that parts other than the electrode part of a high intensity | strength metal needle are insulated with resin.
本発明の電極円錐形先端部の直径は、脳内への刺入性、測定物質との反応性の点から、０．０１〜０．３ｍｍであり、特に０．１〜０．２ｍｍが好ましい。 The diameter of the tip of the electrode cone of the present invention is 0.01 to 0.3 mm, particularly preferably 0.1 to 0.2 mm, from the viewpoint of penetration into the brain and reactivity with the measurement substance. .
本発明の電極は、高強度金属針の先端部長さ０．０２〜２．０ｍｍ部分が高ホウ素原料混入（原料仕込みとして３〜８％ホウ素原料）ダイヤモンドで蒸着されている。原料ホウ素原料混入量が３％未満では、測定感度が十分でない。より好ましいホウ素原料混入率は４〜８％であり、特に好ましくは５％程度である。 In the electrode of the present invention, the tip portion of the high-strength metal needle having a length of 0.02 to 2.0 mm is vapor-deposited with high boron raw material mixed (3 to 8% boron raw material as raw material preparation) diamond. If the raw material boron raw material mixing amount is less than 3%, the measurement sensitivity is not sufficient. A more preferable boron raw material mixing rate is 4 to 8%, particularly preferably about 5%.
高強度金属針の先端部へのホウ素原料混入ダイヤモンドの蒸着処理は、７００〜９００℃で２〜１２時間行えばよい。本発明においては、まずタングステンへの接着性に優れる０．５〜２％低ホウ素原料混入ダイヤモンドで蒸着し、その表面にさらに高感度の３〜８％高ホウ素原料混入ダイヤモンドで蒸着するのが、高強度と高感度の両者を達成するうえで、特に好ましい。タングステン面から電極表面へのホウ素濃度の上昇は二段階、多段階、あるいは連続的でもよい。さらに、まず０．５〜１．５％ホウ素原料混入ダイヤモンドで蒸着し、その表面にさらに４〜６％ホウ素原料混入ダイヤモンドで蒸着するのが特に好ましい。 The vapor deposition treatment of the boron raw material mixed diamond on the tip portion of the high-strength metal needle may be performed at 700 to 900 ° C. for 2 to 12 hours. In the present invention, it is first deposited with 0.5 to 2% low boron raw material mixed diamond excellent in adhesion to tungsten, and further deposited on the surface with 3 to 8% high boron raw material mixed diamond. It is particularly preferable for achieving both high strength and high sensitivity. The increase in boron concentration from the tungsten surface to the electrode surface may be two-step, multi-step, or continuous. Further, it is particularly preferable to deposit with 0.5 to 1.5% boron raw material mixed diamond and further to the surface with 4 to 6% boron raw material mixed diamond.
このような二重、段階的あるいは連続的にホウ素濃度を高めた蒸着処理により、金属表面に均一に高ホウ素混入ダイヤモンドが蒸着できるが、さらに感度を向上させ、正確な測定を可能とするため、内部金属の露出部分を樹脂の点状塗布により充填するのが好ましい。前記蒸着処理により点状に金属が露出することがあり、かかる露出部の樹脂塗布硬化により、点状に絶縁部が生じるが、測定感度は向上する。 By such a double, stepwise or continuous vapor deposition process, the boron concentration can be uniformly deposited on the metal surface, but the sensitivity is further improved and accurate measurement is possible. It is preferable to fill the exposed portion of the internal metal with a dot application of resin. Although the metal may be exposed in the form of dots due to the vapor deposition treatment, an insulating part is generated in the form of dots due to the resin coating and curing of the exposed parts, but the measurement sensitivity is improved.
本発明電極における電極部以外の部分は、樹脂塗布により絶縁するのが、操作性、測定感度の点で好ましい。絶縁に用いられる樹脂は、特に制限されないが、エポキシ樹脂、カシュー塗料、パリレン樹脂等が好ましい。電極部以外の部分は、前記高強度金属でもよいし、さらに表面を絶縁したステンレス管、銅線を接続して用いてもよい。電極全体の長さは、延長管の着脱により調節可能で、マニピュレーターにおける操作性の点から１０〜２０ｃｍ程度に延長可能であることが好ましい。 In terms of operability and measurement sensitivity, it is preferable to insulate portions other than the electrode portion in the electrode of the present invention by resin coating. The resin used for insulation is not particularly limited, but epoxy resin, cashew paint, parylene resin and the like are preferable. The portion other than the electrode portion may be the high-strength metal, or may be used by connecting a stainless steel tube or a copper wire whose surface is further insulated. The length of the entire electrode can be adjusted by attaching and detaching the extension tube, and is preferably extendable to about 10 to 20 cm from the viewpoint of operability in the manipulator.
本発明の微小電極を用いれば、組織を侵襲することなく、脳内に刺入でき、また脳内に留置もできるため、長時間の脳内物質濃度の変動をモニターできる。即可能な脳内物質としては、ドパミン、ノルアドレナリン、セロトニン等の脳内アミンの他、アスコルビン酸、アデノシンが挙げられる。 If the microelectrode of the present invention is used, it can be inserted into the brain without invading the tissue, and can also be placed in the brain, so that fluctuations in the substance concentration in the brain for a long time can be monitored. Examples of intracerebral substances that can be immediately used include amines in the brain such as dopamine, noradrenaline, and serotonin, ascorbic acid, and adenosine.
測定手段としては、本発明電極を脳内に刺入し、参照電極及び対電極を用いたボルタメトリーにより行うのが好ましい。ここで参照電極及び対電極としては銀塩化銀電極、白金線等が用いられる。さらに本発明においては、非特許文献３においてはパルスボルタメトリー法により測定し５０Ｈｚ１００発という強い刺激に対する応答をようやく検出できるものだったが、アンペロメトリー（図４）およびサイクリックボルタメトリー（図５）を利用することで大幅に感度が高まり、これらの手法では高ホウ素の効果とあいまって４０ｎＭのドパミンすら検出可能となっている。電位を一定にした定電位アンペロメトリーにより測定するのがより好ましい。ここで定電位アンペロメトリーとは、銀塩化銀に対し０．５〜１．０Ｖにダイヤモンド電極を保つことによりドパミンの酸化電流を検出する手法である。定電位アンペロメトリーにより、脳内アミンがより高感度で特に高速で測定できる。パルスボルタメトリー、サイクリックボルタメトリーといった電位を変動させる手法であれば、サンプリング頻度においてアンペロメトリーに劣るが分子を選別して測定するのに有利である。 As a measuring means, the electrode of the present invention is preferably inserted into the brain and voltammetry is preferably performed using a reference electrode and a counter electrode. Here reference electrode and a silver-silver chloride electrode as the counter electrode, white gold or the like is used. Further, in the present invention, in Non-Patent Document 3, a response to a strong stimulus of 50 Hz 100 times was finally detected as measured by the pulse voltammetry method, but amperometry (FIG. 4) and cyclic voltammetry (FIG. 5). ) Significantly increases the sensitivity, and even with these effects, even 40 nM dopamine can be detected with these techniques. It is more preferable to measure by constant potential amperometry with a constant potential. Here, the constant potential amperometry is a technique for detecting the oxidation current of dopamine by keeping the diamond electrode at 0.5 to 1.0 V with respect to silver chloride. Constant potential amperometry can measure amines in the brain with higher sensitivity and especially at high speed. A technique that varies the potential, such as pulse voltammetry and cyclic voltammetry, is inferior to amperometry at the sampling frequency, but is advantageous for selecting and measuring molecules.
次に実施例を挙げて本発明を詳細に説明する。 EXAMPLES Next, an Example is given and this invention is demonstrated in detail.
実施例１
直径０．３ｍｍ長さ３ｃｍの細いタングステン針表面に５％ホウ素原料混入ダイヤモンド（ＢＤＤ）を蒸着させ、先端部以外を絶縁した（図１）。まずタングステンロッドを針状とするため先端部分を２Ｍ水酸化ナトリウム（ＮａＯＨ）中３Ｖクロノアンペロメトリ（ＣＡ）により電解研磨し、先端経を約１０〜２０μｍ、テーパー角３．８度とした。その後、酸化皮膜を除去するためにフッ化水素（ＨＦ）中に２０分浸漬した。あるいは同様の形態の加工品（有明マテリアル株式会社）を使用した．タングステン表面におけるダイヤモンド層成長を促進するため、ダイヤモンドパウダーをイソプロパノール中で９０分間核付け処理した。針状に成型した金属針を水素プラズマ中に置き、一方で炭素源及びホウ素源を含む溶液を水素ガスによりバブリングすることで気化させ、これを水素プラズマ中に導入することで表面にダイヤモンド層を形成させる。すなわち、作成したタングステン針をプラズマ装置中（ＡＳＴｅＸ Ｃｏｒｐ．図２）で水素流量３００ｓｓｃｍ、プラズマ出力２５００Ｗ、圧力６０Ｔｏｒｒ、ステージ温度７００〜９００℃にてダイヤモンド層を蒸着した。初め５時間は１％ホウ素仕込み（トリメトキシボラン４．０５ｍＬ／アセトン５０ｍＬ）、続く５時間は５％ホウ素仕込み（トリメトキシボラン１８．５ｍＬ／アセトン４０ｍＬ）で二段生成させた。１％ホウ素時間を短縮した場合はアンペロメトリーに使用可能な電極ができなかった（表１）。ＢＤＤ微小電極を作成した先端１ｍｍ以外をエポキシ樹脂及びカシュー塗料を用いて絶縁し、安定した電気化学センサーとして使用できる形状に成型した（図１）。先端から１．０ｍｍの範囲を除いてエポキシ樹脂（セメダイン１５６５）を塗布し先端を電極として確保した。ダイヤモンド層には多くの場合微小な穴があり、タングステン面が露出して電気化学計測に大きな障害となる。ダイヤモンド剥離部分を実体顕微鏡下に探索し、剥離部分にエポキシ樹脂を点状に塗布し絶縁した。
Example 1
5% boron raw material mixed diamond (BDD) was vapor-deposited on the surface of a thin tungsten needle having a diameter of 0.3 mm and a length of 3 cm to insulate other than the tip (FIG. 1). First, in order to make the tungsten rod into a needle shape, the tip portion was electrolytically polished by 3V chronoamperometry (CA) in 2M sodium hydroxide (NaOH), and the tip length was about 10 to 20 μm, and the taper angle was 3.8 degrees. Thereafter, it was immersed in hydrogen fluoride (HF) for 20 minutes in order to remove the oxide film. Alternatively, a similar processed product (Ariake Material Co., Ltd.) was used. Diamond powder was nucleated in isopropanol for 90 minutes to promote diamond layer growth on the tungsten surface. A metal needle shaped like a needle is placed in a hydrogen plasma, while a solution containing a carbon source and a boron source is vaporized by bubbling with hydrogen gas, and this is introduced into the hydrogen plasma to form a diamond layer on the surface. Let it form. That is, a diamond layer was vapor-deposited in a plasma apparatus (ASTeX Corp. FIG. 2) with a hydrogen flow rate of 300 sscm, a plasma output of 2500 W, a pressure of 60 Torr, and a stage temperature of 700 to 900 ° C. The first 5 hours were charged with 1% boron (trimethoxyborane 4.05 mL / acetone 50 mL), and the next 5 hours with 5% boron charge (trimethoxyborane 18.5 mL / acetone 40 mL). When 1% boron time was shortened, an electrode usable for amperometry was not obtained (Table 1). Except for the tip of 1 mm on which the BDD microelectrode was formed, the epoxy resin and cashew paint were used to insulate and form into a shape that could be used as a stable electrochemical sensor (FIG. 1). Except for a range of 1.0 mm from the tip, epoxy resin (Cemedine 1565) was applied to secure the tip as an electrode. In many cases, the diamond layer has minute holes, and the tungsten surface is exposed, which is a major obstacle to electrochemical measurement. The diamond peeling portion was searched under a stereomicroscope, and an epoxy resin was applied to the peeling portion in the form of dots to insulate.
軸部の絶縁処理は、当初は外径０．７５ｍｍのガラス管にダイヤモンド針を入れて隙間をエポキシ樹脂で封入した（図１上）。さらに細く仕上げるために、ＢＤＤ針をアセトンにて脱脂の後カシュー塗料による絶縁を試み、好成績を得た（図１下）。サル用マニピュレーターにて操作可能な長さを得るために、２４Ｇステンレス管に接続し圧着、全長１５ｃｍに加工したうえで電気的に導通を確保した。タングステン針及びステンレス管軸部にカシュー塗料を塗布し絶縁した。カシュー塗料のみでは脳組織との摩擦が生じる先端部の強度が充分でなく、またエポキシ樹脂は滴状になりやすく長い軸に均一に塗布するのは困難であった（表１）。 Insulation treatment of the shaft portion was initially performed by inserting a diamond needle into a glass tube having an outer diameter of 0.75 mm and sealing the gap with an epoxy resin (upper part of FIG. 1). In order to achieve a finer finish, the BDD needle was degreased with acetone and then insulated with a cashew paint, and good results were obtained (bottom of FIG. 1). In order to obtain a length operable with a monkey manipulator, it was connected to a 24G stainless steel tube, crimped, processed to a total length of 15 cm, and electrically connected. Cashew paint was applied to the tungsten needle and stainless steel tube shaft for insulation. The cashew paint alone does not provide sufficient strength at the tip where friction with the brain tissue occurs, and the epoxy resin tends to form droplets, making it difficult to uniformly apply to the long shaft (Table 1).
実施例２
ＢＤＤ微小電極は中性燐酸緩衝液（ＰＢＳ）中のドパミンを高感度で検出可能であった（図３、４、５）。銀塩化銀電極を参照電極及び対電極とし、電極を＋６００ｍＶに保持した定電位アンペロメトリーを用いた（図３、４）。図３では、中性燐酸緩衝液（ＰＢＳ）が流れる噴水中に電極を浸漬しポテンショスタット及び記録用コンピュータに接続し電気化学測定を行い、試験化合物を含むＰＢＳ液に流路を切り替えることができる。図３にアンペロメトリー法によるｉｎ−ｖｉｔｒｏでのドパミン検出試験結果を示す。ドパミンを４０、２００、１０００ｎＭ含む液に順次切り替え、測定電流の変動を測定した（図４）。また、反応分子の電位依存性を同定可能なサイクリックボルタメトリー（−０．２Ｖ ｔｏ １．５Ｖ、４００Ｖ／ｓ）による検出も可能であった（図５）。図５では、ドパミンを４０、２００、１０００ｎＭ含む液に順次切り替え、測定電流の変動を測定した。
Example 2
The BDD microelectrode was able to detect dopamine in neutral phosphate buffer (PBS) with high sensitivity (FIGS. 3, 4, and 5). Constant-potential amperometry was used in which the silver-silver chloride electrode was used as a reference electrode and a counter electrode and the electrode was held at +600 mV (FIGS. 3 and 4). In FIG. 3, an electrode is immersed in a fountain in which a neutral phosphate buffer (PBS) flows, and connected to a potentiostat and a recording computer to perform electrochemical measurement, and the flow path can be switched to a PBS solution containing a test compound. . FIG. 3 shows the results of an in-vitro dopamine detection test by amperometry. Changes in measurement current were measured by sequentially switching to solutions containing 40, 200, and 1000 nM dopamine (FIG. 4). Moreover, the detection by the cyclic voltammetry (-0.2V to 1.5V, 400V / s) which can identify the voltage dependence of the reaction molecule | numerator was also possible (FIG. 5). In FIG. 5, the change of the measurement current was measured by sequentially switching to solutions containing 40, 200, and 1000 nM dopamine.
実施例３
同一の微小電極の部分を段階的に絶縁塗装して比較した。
２本の５％ホウ素混入微小電極の結果の平均を表２及び図６に示す。
０．６Ｖ定電位アンペロメトリー及びＦＳＣＶ（−０．２ ｔｏ １．５Ｖ）により１０００ｎＭドパミンによる振幅のその直前のノイズ標準偏差ＳＤに対する比率をＳ／Ｎ比とした。
良好なＳ／Ｎ比を得るためには長さ０．３ｍｍ以上必要であった反面、電極露出部が長いと流速変動に対するノイズ電流が生じやすい性質が見られた。従って、電極長は０．２〜２．０ｍｍ、特に０．３〜０．７ｍｍが好ましいことがわかった。
Example 3
The same microelectrode part was insulatively coated in stages and compared.
The average of the results of two 5% boron-containing microelectrodes is shown in Table 2 and FIG.
The ratio of the amplitude due to 1000 nM dopamine to the noise standard deviation SD immediately before was defined as the S / N ratio by 0.6 V constant potential amperometry and FSCV (−0.2 to 1.5 V).
In order to obtain a good S / N ratio, a length of 0.3 mm or more was necessary. On the other hand, when the electrode exposed part was long, a characteristic that a noise current with respect to fluctuations in flow rate was likely to occur was observed. Accordingly, it has been found that the electrode length is preferably 0.2 to 2.0 mm, particularly preferably 0.3 to 0.7 mm.
実施例４
脳内での測定が可能であるか検証するために、本電極をマウス脳線条体に刺入し電気刺激によりドパミン放出を生じさせたところ、それをリアルタイムで検出可能であった（図７、表３）。図７では、ドパミン神経路を電気刺激した際の線条体における電流変動を示す。横軸０秒の時点から３０Ｈｚ１２発電気刺激を与えた。表３では、ドパミン神経路（ＭＦＢ）を３０Ｈｚ刺激パルス数による検出電流をＳ／Ｎ比で示し、ダイヤモンド電極アンペロメトリー（ＢＤＤ−ａｍｐｅｒｏ）及び同時に行った従来法カーボンファイバーによるサイクリックボルタメトリー法（ｃｆ−ＦＳＣＶ）と比較した。
Example 4
In order to verify whether measurement in the brain is possible, when this electrode was inserted into the mouse striatum and dopamine was released by electrical stimulation, it could be detected in real time (FIG. 7). Table 3). FIG. 7 shows current fluctuations in the striatum when the dopamine nerve tract is electrically stimulated. From the time point of 0 sec on the horizontal axis, 12 Hz electrical stimulation was applied. Table 3 shows dopamine nerve tract (MFB) detected current by 30 Hz stimulation pulse number in S / N ratio, diamond electrode amperometry (BDD-ampero) and cyclic voltammetry with conventional carbon fiber performed simultaneously. Comparison with (cf-FSCV).
実施例５
大型脳における利用を検証するために、サル線条体に刺入し（図８）ジュースを報酬として中脳ドパミン神経を活動せしめたところ（Schultz W.Behavioral dopamine signals.Trends Neurosci. 2007 May;30(5):203-10.）、報酬予告信号後のドパミン放出を検出可能であった（図９）。
Example 5
In order to verify the use in the large brain, the monkey striatum was inserted (Fig. 8) and the midbrain dopamine nerve was activated using juice as a reward (Schultz W. Behavioral dopamine signals. Trends Neurosci. 2007 May; 30 (5): 203-10.), It was possible to detect the release of dopamine after the reward notice signal (FIG. 9).
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| JP2015039544A (en) * | 2013-08-22 | 2015-03-02 | 学校法人慶應義塾 | METHOD AND DEVICE FOR MEASURING INTRAVITAL pH USING DIAMOND MICROELECTRODE |
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