JPH09292395A - Measuring method for microregion and microcarrier using it - Google Patents
Measuring method for microregion and microcarrier using itInfo
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- JPH09292395A JPH09292395A JP12933096A JP12933096A JPH09292395A JP H09292395 A JPH09292395 A JP H09292395A JP 12933096 A JP12933096 A JP 12933096A JP 12933096 A JP12933096 A JP 12933096A JP H09292395 A JPH09292395 A JP H09292395A
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- Prior art keywords
- microcarrier
- antibody
- modified
- spheres
- modification
- Prior art date
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、微小領域の物理化
学的情報を光学的に検出する技術に関するものである。TECHNICAL FIELD The present invention relates to a technique for optically detecting physicochemical information in a minute region.
【０００２】[0002]
【従来の技術】微小体の配向により物理的情報を検出す
る従来の技術として、液晶を利用した電場の検出、常磁
性体粉末を利用した磁場の検出が挙げられる。前者を半
導体集積回路に応用することにより、接続配線間の電磁
気的干渉に関する情報が得られ、回路の設計向上に役立
つ。また後者の技術を、磁気記録媒体に応用することに
より、記録密度の高度化に必要な磁区に関する情報が得
られる。それぞれ、微小領域の物理量を二次元的に可視
化することに特徴がある。2. Description of the Related Art Conventional techniques for detecting physical information based on the orientation of microscopic bodies include detection of an electric field using liquid crystals and detection of a magnetic field using paramagnetic powder. By applying the former to a semiconductor integrated circuit, information on electromagnetic interference between connection wirings can be obtained, which is useful for improving the circuit design. Further, by applying the latter technique to a magnetic recording medium, it is possible to obtain information on magnetic domains necessary for increasing the recording density. Each is characterized by two-dimensionally visualizing the physical quantity of a minute area.
【０００３】一方、微小体の配向による化学的情報の検
出技術として、蛍光色素で修飾された抗体の利用が挙げ
られる。抗原抗体反応に伴い蛍光色素の回転速度が変化
するため、蛍光偏向解消法により反応を検出、すなわち
抗原の有無を判断できる。細胞の測定に応用すれば、細
胞内の抗原分布の情報が得られ、細胞に基づく健康診断
が可能になる。On the other hand, as a technique for detecting chemical information based on the orientation of microscopic bodies, use of an antibody modified with a fluorescent dye can be mentioned. Since the rotation speed of the fluorescent dye changes with the antigen-antibody reaction, the reaction can be detected, that is, the presence or absence of the antigen can be determined by the fluorescence polarization elimination method. When applied to the measurement of cells, information on the distribution of antigens in cells can be obtained, and cell-based medical examinations can be performed.
【０００４】[0004]
【発明が解決しようとする課題】微小体の配向による物
理化学的情報の光学的測定においては、環境に反応して
微小体が配向し、しかも配向を光学的に検出することが
必要である。この二つの機能を一つの微小体に持たせる
ことはしばし困難である。In the optical measurement of physicochemical information based on the orientation of fine particles, it is necessary to orient the fine particles in response to the environment and to detect the orientation optically. It is often difficult to give these two functions to one micro body.
【０００５】本発明は、機能性微小体の設計、製造を容
易にし、しかも複数の物理化学的情報を同時に計測する
のに必要な多機能化さえも容易に実現するのが目的であ
る。It is an object of the present invention to facilitate the design and manufacture of functional microparticles, and also to easily realize the multi-functionalization necessary for simultaneously measuring a plurality of physicochemical information.
【０００６】[0006]
【課題を解決するための手段】環境に反応する機能と配
向の検出に必要な標識といった異なった機能を別々な分
子に持たせ、それぞれの分子を微小担体の表面に結合さ
せる。この際、分子を機能別に限定された領域、しかも
相対位置が明確な領域に選択的に結合する必要が生じ
る。その手段として、微小担体表面に蒸着による結合の
下地造りを行う。蒸着により、担体の片面のみに形成さ
れる薄膜を分子結合の下地として用いると、微小担体の
限定領域における結合が可能となる。また、蒸着薄膜が
施されていない表面に対して、別な分子を選択的に結合
することにより、二種類の分子をそれぞれ、異なった限
定領域に結合することができる。さらに、微小担体の蒸
着を異なった方向から異なった材料で行うことにより、
３つ以上の分子も選択的に結合できる。[Means for Solving the Problems] Different molecules have different functions such as a function of reacting to the environment and a label necessary for detecting orientation, and each molecule is bound to the surface of a microcarrier. In this case, it becomes necessary to selectively bind the molecule to a region defined by function and a region having a clear relative position. As a means therefor, a bonding underlayer is formed on the surface of the microcarrier by vapor deposition. When a thin film formed on only one side of the carrier by vapor deposition is used as a base for molecular bonding, bonding in a limited area of the microcarrier becomes possible. Further, by selectively binding different molecules to the surface on which the vapor-deposited thin film is not applied, it is possible to bind two kinds of molecules to different restricted regions. Furthermore, by depositing microcarriers from different directions and with different materials,
More than two molecules can also be selectively bound.
【０００７】[0007]
【発明の実施の形態】異なった形状の修飾化微小担体を
図１に示す。同図（ａ）は片面のみ修飾された球状担体
１、（ｂ）はディスク状担体２、また（ｃ）は面ごとに
異なる修飾面（Ａ、Ｂ、Ｃ）３、４、５を有する立方体
状担体６、（ｄ）は両端に異なる修飾領域（Ａ、Ｂ）
７、８を有する棒状担体９である。図１１の写真は、球
状担体１の実例を示す。DETAILED DESCRIPTION OF THE INVENTION Different shapes of modified microcarriers are shown in FIG. In the figure, (a) is a spherical carrier 1 modified on only one side, (b) is a disk-shaped carrier 2, and (c) is a cube having modified surfaces (A, B, C) 3, 4, 5 which are different for each surface. Shaped carrier 6, (d) has different modified regions (A, B) at both ends
It is a rod-shaped carrier 9 having 7 and 8. The photograph in FIG. 11 shows an example of the spherical carrier 1.
【０００８】球状担体を用いた際における、不均一修飾
方法の概念を図２に示す。同図（ａ）に示すように、ま
ず基板１０上に置かれた未修飾微小担体１１に金、銀、
銅、プラチナなどの金属またはシリコンなどの蒸着材料
１２を蒸着することにより、担体の片面のみ薄膜１３を
形成し、カップリング処理により薄膜１３上に結合基を
有する微小担体を調製した。次に同図（ｂ）のように、
抗体、抗原、アビジン、ビオチン、プロティンＡ、プロ
ティンＧ、ペプチド、磁性体、エレクトレット、色素、
蛍光色素、放射性同位元素など１４を結合基にカップリ
ングすることにより、片面のみ修飾された微小担体１５
を得た。FIG. 2 shows the concept of the method of heterogeneous modification when a spherical carrier is used. As shown in FIG. 3A, first, gold, silver,
A thin film 13 was formed on only one surface of the carrier by depositing a deposition material 12 such as a metal such as copper or platinum or silicon, and a microcarrier having a bonding group on the thin film 13 was prepared by a coupling treatment. Next, as shown in FIG.
Antibody, antigen, avidin, biotin, protein A, protein G, peptide, magnetic substance, electret, dye,
A microcarrier 15 modified on only one side by coupling 14 such as a fluorescent dye or a radioisotope to a binding group.
I got
【０００９】一方、同図（ｃ）のように、あらかじめ抗
体、抗原、アビジン、ビオチン、プロティンＡ、プロテ
ィンＧ、ペプチド、磁性体、エレクトレット、色素、蛍
光色素などで表面を全面修飾された微小担体１６を基板
１０上に置き、同図（ｄ）のように、蒸着または電子、
中性子などのビ−ム、Ｘ線、紫外線などの照射１７によ
り微小担体上に修飾機能損失面１８を片面のみ形成する
こともできた。On the other hand, as shown in FIG. 1C, a microcarrier whose surface is preliminarily modified with an antibody, an antigen, avidin, biotin, protein A, protein G, peptide, magnetic substance, electret, dye, fluorescent dye, etc. 16 is placed on the substrate 10 and, as shown in FIG.
It was also possible to form only one modified functional loss surface 18 on the microcarrier by irradiation 17 with beams such as neutrons, X-rays, and ultraviolet rays.
【００１０】（実施例１）実施例１における微小担体製
造方法を図３に示す。同図（ａ）に示すように、蛍光色
素を含有する直径 0.02〜100 ミクロメ−トルのポリス
チレン球２０をガラス、シリコンなどの平坦度の高い基
板２１上に重ならない様に分散した。同図（ｂ）のよう
に、基板を蒸着装置の中に設置し、真空中で金２２を基
板上の球に蒸着し、球の片面に 10〜100 ナノメ-トルの
金薄膜２３を形成した。蒸着後、エタノ−ル、テトラヒ
ドロフラン、シクロヘキサンなどの溶媒に溶かしたジチ
オ−ルの混合液にポリスチレン球を添加し、同図（ｃ）
のように金薄膜２３表面のみにジチオ−ル分子層２４を
形成した。次に、同図（ｄ）のようにコロイド金、アル
キルハライド、マレイミド、アジリヂンなどで修飾され
たスルフヒドリル基と結合する抗体２５を添加し、球の
金薄膜表面を修飾した。その結果、片面のみが抗体で修
飾された球が得られた。(Example 1) FIG. 3 shows a method for producing a microcarrier in Example 1. As shown in FIG. 3A, polystyrene spheres 20 containing a fluorescent dye and having a diameter of 0.02 to 100 micrometer were dispersed on a substrate 21 having high flatness such as glass or silicon so as not to overlap. As shown in FIG. 6B, the substrate was placed in a vapor deposition apparatus, and gold 22 was vapor-deposited on a sphere on the substrate in vacuum to form a gold thin film 23 of 10 to 100 nanometer on one side of the sphere. . After vapor deposition, polystyrene spheres were added to a mixed solution of dithiol dissolved in a solvent such as ethanol, tetrahydrofuran or cyclohexane, and the same figure (c).
As described above, the dithiol molecular layer 24 was formed only on the surface of the gold thin film 23. Next, as shown in FIG. 3D, an antibody 25 that binds to a sulfhydryl group modified with colloidal gold, alkyl halide, maleimide, aziridin, etc. was added to modify the gold thin film surface of the sphere. As a result, spheres having only one surface modified with the antibody were obtained.
【００１１】細胞表面の膜蛋白質の検出に用いた測定例
を図４に示す。膜蛋白質３０を抗原とする抗体２６を有
する微小担体が、抗原抗体反応によりリン脂質二重膜３
１表面に結合した。蛍光は金薄膜２３に妨げられるた
め、結合されてない微小担体を洗い流すと、蛍光３２は
結合面とは逆の面のみより観察された。したがって、蛍
光観察により抗原抗体反応だけではなく、抗原抗体結合
面の配向情報を得ることができた。FIG. 4 shows a measurement example used for detecting a membrane protein on the cell surface. A microcarrier having an antibody 26 having a membrane protein 30 as an antigen is converted into a phospholipid bilayer membrane 3 by an antigen-antibody reaction.
Bound to one surface. Since the fluorescence is blocked by the gold thin film 23, when the unbound microcarriers were washed away, the fluorescence 32 was observed only from the surface opposite to the bonding surface. Therefore, not only the antigen-antibody reaction but also the orientation information of the antigen-antibody binding surface could be obtained by the fluorescence observation.
【００１２】（実施例２）実施例２におけるポリスチレ
ン球の修飾方法を図５に示す。同図（ａ）に示すよう
に、まずアミン４０で修飾された表面を有する直径 0.0
2〜100 ミクロメ−トルのポリスチレン球４１をガラ
ス、シリコンなどの平坦度の高い基板上４２に重ならな
い様に分散した。次に同図（ｂ）のように、基板を蒸着
装置の中に設置し、真空中で銀４３を基板上の球４１に
蒸着し、球の片面に 1〜100 ナノメ−トルの銀薄膜４４
を形成した。銀薄膜４４の酸化後、同図（ｃ）のように
スクシンイミドエステル、イソチオシアン酸塩、塩化ス
ルホン、ＮＢＤハライド、ジクロロトリアジンなどを有
しアミンと結合する抗体４５で、蒸着されていない表面
４６のみを修飾した。さらに、同図（ｄ）のようにカル
ボキシル基を有する蛍光色素４７を微小担体に添加する
ことにより、酸化銀４８の表面を蛍光色素４７で修飾し
た。これにより同図（ｅ）のように、片面は抗体４５、
もう片面は蛍光色素４７というように二種類の物質で修
飾されたポリスチレン球４９が得られた。ポリスチレン
球４９は電荷５０を帯びており、抗体４５で修飾された
表面と蛍光色素４７で修飾された表面は異なる電荷５１
を帯びる結果、担体全体として電気双極子モ−メント５
２を有する。Example 2 A method for modifying polystyrene spheres in Example 2 is shown in FIG. As shown in FIG. 3A, a diameter of 0.0 having a surface modified with amine 40 is first obtained.
Polystyrene spheres 41 of 2 to 100 micrometer were dispersed on a substrate 42 having high flatness such as glass or silicon so as not to overlap. Next, as shown in FIG. 2B, the substrate is placed in a vapor deposition apparatus, silver 43 is vapor-deposited on the sphere 41 on the substrate in a vacuum, and a silver thin film 44 of 1 to 100 nanometer is formed on one side of the sphere.
Was formed. After the oxidation of the silver thin film 44, as shown in FIG. 7C, the antibody 45 having succinimide ester, isothiocyanate, sulfone chloride, NBD halide, dichlorotriazine, etc., which binds to the amine, is used to expose only the surface 46 which is not deposited Qualified. Further, the surface of silver oxide 48 was modified with fluorescent dye 47 by adding fluorescent dye 47 having a carboxyl group to the microcarrier as shown in FIG. As a result, as shown in (e) of FIG.
Polystyrene spheres 49 modified with two kinds of substances such as fluorescent dye 47 on the other side were obtained. The polystyrene sphere 49 has a charge 50, and the surface modified with the antibody 45 and the surface modified with the fluorescent dye 47 have different charges 51.
As a result, the carrier as a whole has an electric dipole moment 5.
2
【００１３】電場下におけるポリスチレン球４９の配向
を図６に示す。電場６０により電気双極子モ−メントを
有する球４９を配向させることができた。担体表面上の
抗体４５が抗原６１と反応すると、抗原の電荷６２によ
り担体全体の電気双極子モ−メントが変化した。したが
って、電場下での担体の配向速度に変化が生じ、球４９
の片面のみから観察される蛍光６３により配向を検出で
きた。The orientation of polystyrene spheres 49 under an electric field is shown in FIG. The electric field 60 was able to orient the sphere 49 with the electric dipole moment. When the antibody 45 on the surface of the carrier reacts with the antigen 61, the electric charge 62 of the antigen changes the electric dipole moment of the entire carrier. Therefore, a change occurs in the orientation speed of the carrier under the electric field, and the sphere 49
The orientation could be detected by the fluorescence 63 observed from only one surface of the.
【００１４】免疫計測方法を図７に示す。同図（ａ）の
ように、一対の電極７０を有するセル７１中の被検体液
７２にポリスチレン球４９を加えた。電極７０に変調電
位７３を印加することにより、球４９を周期的に再配向
できる。配向を検出するためには、光源７４で球４９の
蛍光色素を励起し、励起光をカットオフする光学フィル
タ−７５を有する光センサ−７６にて蛍光信号のみを検
出した。球４９の向きに依存して蛍光信号７７が振幅７
８で変化した。変調電位７３の周波数に依存して、蛍光
信号７７の振幅７８が変化する様子を同図（ｂ）の特性
曲線７９で示す。低周波領域では球４９は電場変調に追
従できるが、カットオフ周波数８０より高周波領域では
追従できなくなり、蛍光信号７７の振幅７８が特性曲線
７９で示される様に低下した。カットオフ周波数は球４
９の電気双極子モ−メントに依存するため、球４９表面
の抗体４５と反応する抗原６１が被検体液７２中に含ま
れる際、カットオフ周波数が変わった。例えば、抗原抗
体反応の結果、特性曲線８１が示す様にカットオフ周波
数が高周波側８２に移動した。また場合に依っては低周
波側に移動した。したがって、球４９からの蛍光信号７
７の振幅７８の変調電位周波数依存性を測定することに
より、抗原抗体反応を検出できた。The immunoassay method is shown in FIG. As shown in FIG. 6A, polystyrene spheres 49 were added to the sample liquid 72 in the cell 71 having the pair of electrodes 70. By applying a modulation potential 73 to the electrode 70, the sphere 49 can be periodically reoriented. In order to detect the orientation, the fluorescent dye of the sphere 49 was excited by the light source 74, and only the fluorescent signal was detected by the optical sensor-76 having the optical filter-75 for cutting off the excitation light. The fluorescence signal 77 has an amplitude of 7 depending on the direction of the sphere 49.
Changed by 8. A characteristic curve 79 in FIG. 7B shows how the amplitude 78 of the fluorescence signal 77 changes depending on the frequency of the modulation potential 73. Although the sphere 49 can follow the electric field modulation in the low frequency region, it cannot follow the high frequency region beyond the cutoff frequency 80, and the amplitude 78 of the fluorescence signal 77 is lowered as shown by the characteristic curve 79. Cutoff frequency is sphere 4
Since it depends on the electric dipole moment of 9, the cutoff frequency was changed when the antigen 61 that reacts with the antibody 45 on the surface of the sphere 49 was contained in the sample liquid 72. For example, as a result of the antigen-antibody reaction, the cutoff frequency moved to the high frequency side 82 as shown by the characteristic curve 81. In some cases, it moved to the low frequency side. Therefore, the fluorescence signal 7 from the sphere 49
The antigen-antibody reaction could be detected by measuring the modulation potential frequency dependence of the amplitude 78 of 7.
【００１５】（実施例３）フォトリトグラフィ−の技術
を用いた酸化シリコン微小担体の製造方法を図８に示し
た。まず同図（ａ）に示すように、シリコン基板９０の
上に酸化シリコン９１とポジ型フォトレジスト９２の薄
膜を形成した。光マスク９３によりディスク形状のパタ
−ンをポジ型フォトレジスト９２に投影し、同図（ｂ）
のように照射領域９４のフォトレジストを現像により溶
解除去した。次に同図（ｃ）のようにＣＨＦ３ガス９５
を用いたプラズマエッチングにより酸化シリコン薄膜９
６を加工した。未現像のポジ型フォトレジスト９７を取
り除き、シリコン基板９０のＫＯＨ湿式エッチングによ
り、同図（ｄ）のように酸化シリコン微小担体９８をエ
ッチングされたシリコン基板９９から剥離、回収した。(Embodiment 3) FIG. 8 shows a method for producing silicon oxide microcarriers using the photolithography technique. First, as shown in FIG. 9A, a thin film of silicon oxide 91 and a positive photoresist 92 was formed on a silicon substrate 90. A disk-shaped pattern is projected on the positive photoresist 92 by the optical mask 93, and the same figure (b) is shown.
As described above, the photoresist in the irradiation region 94 was dissolved and removed by development. Next, as shown in FIG.
Silicon oxide thin film 9 by plasma etching using
6 was processed. The undeveloped positive photoresist 97 was removed, and the silicon oxide microcarriers 98 were peeled and collected from the etched silicon substrate 99 by KOH wet etching of the silicon substrate 90 as shown in FIG.
【００１６】酸化シリコン微小担体の修飾方法を図９に
示す。まず同図（ａ）のように、酸化シリコン微小担体
９８を重ならない様に基板１００上に置き、アルミニウ
ム１０１を蒸着した。蒸着後、同図（ｂ）のようにアル
ミニウムの酸化によりＯＨ基層１０２を形成し、シリコ
ン面にはシランカップリング処理によりスルフヒドリル
基層１０３を形成した。次に、同図（ｃ）のようにＣＯ
ＯＨ基を有するフルオレセイン、イオシン、エリトロシ
ン系の蛍光色素（Ａ）１０４のエステル化反応によりＯ
Ｈ基層１０２を修飾し、スルフヒドリル基層１０３をマ
レイミド基を有するフルオレセイン、イオシン、エリト
ロシン系の蛍光色素（Ｂ）１０５で修飾した結果、二種
類の蛍光色素で修飾された酸化シリコン微小担体１０６
が得られた。蛍光色素（Ａ）１０４と蛍光色素（Ｂ）１
０５の蛍光スペクトルはそれぞれ同図（ｄ）に示す１０
７と１０８であり、しかも異なったｐＨ依存性を有す
る。例えば、蛍光色素（Ａ）１０４のｐＫ（Ａ）は蛍光
色素（Ｂ）１０５のｐＫ（Ｂ）より大きくなる様に選択
した。A method for modifying the silicon oxide microcarriers is shown in FIG. First, as shown in FIG. 3A, the silicon oxide microcarriers 98 were placed on the substrate 100 so as not to overlap each other, and aluminum 101 was vapor-deposited. After vapor deposition, an OH base layer 102 was formed by oxidizing aluminum as shown in FIG. 2B, and a sulfhydryl base layer 103 was formed on the silicon surface by a silane coupling treatment. Next, as shown in FIG.
O by the esterification reaction of the fluorescein, iocin, and erythrosine-based fluorescent dye (A) 104 having an OH group
As a result of modifying the H base layer 102 and modifying the sulfhydryl base layer 103 with a fluorescent dye (B) 105 of a maleimide group-containing fluorescein, iocin, or erythrosine group, a silicon oxide microcarrier 106 modified with two types of fluorescent dyes.
was gotten. Fluorescent dye (A) 104 and fluorescent dye (B) 1
The fluorescence spectra of 05 are shown in FIG.
7 and 108, and have different pH dependences. For example, the pK (A) of the fluorescent dye (A) 104 was selected to be larger than the pK (B) of the fluorescent dye (B) 105.
【００１７】図１０（ａ）〜（ｆ）に微小担体の光学、
電気特性のｐＨ依存性を示す。溶液中では微小担体がラ
ンダムに配向しているため、ｐＨ＜ｐＫ（Ｂ）では蛍光
スペクトルは１１０、ｐＫ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）で
は１１１となり、またｐＫ（Ａ）＜ｐＨでは１１２の様
に蛍光が検出されなかった。また、ｐＨによって蛍光色
素の電荷が変化するので、上の三つｐＨ領域によって微
小担体１０６の電気双極子モ−メントが異なった。例え
ば、ｐＫ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）の時に電気双極子モ
−メント１１３が最大値を取る様になる。したがって、
ある特定ｐＨでのみ微小担体が電場に反応して配向し
た。また、微小担体の蛍光スペクトルは面の方向に依存
するので、蛍光スペクトルを観測することにより、電場
下の担体の配向が判る。したがって、蛍光スペクトルか
らｐＨと電場に関する情報が同時に得られる。FIGS. 10 (a) to 10 (f) show the optics of microcarriers,
The pH dependence of electrical properties is shown. Since the microcarriers are randomly oriented in the solution, the fluorescence spectrum is 110 at pH <pK (B), 111 at pK (B) <pH <pK (A), and 112 at pK (A) <pH. No fluorescence was detected as in. Moreover, since the electric charge of the fluorescent dye changes depending on the pH, the electric dipole moment of the microcarrier 106 differs depending on the above three pH regions. For example, when pK (B) <pH <pK (A), the electric dipole moment 113 takes the maximum value. Therefore,
The microcarriers were oriented in response to an electric field only at a certain pH. Moreover, since the fluorescence spectrum of the microcarriers depends on the direction of the plane, the orientation of the carrier under the electric field can be known by observing the fluorescence spectrum. Therefore, information on pH and electric field can be obtained simultaneously from the fluorescence spectrum.
【００１８】[0018]
【発明の効果】本発明により、微小領域の物理化学的情
報を同時に複数、しかも容易に可視化できる。According to the present invention, it is possible to easily visualize a plurality of physicochemical information in a minute area simultaneously.
【図１】本発明により製造、使用される不均一的修飾ま
たは異方性形状微小担体の例を示す図。FIG. 1 is a diagram showing an example of a heterogeneous modified or anisotropic shaped microcarrier produced and used according to the present invention.
【図２】蒸着による微小担体の不均一的修飾方法の概念
図。FIG. 2 is a conceptual diagram of a method of nonuniformly modifying a microcarrier by vapor deposition.
【図３】片面のみに金蒸着されたポリスチレン球に、ジ
チオ−ルを介して抗体を結合させる方法を示す図。FIG. 3 is a view showing a method of binding an antibody to polystyrene spheres having gold vapor-deposited on only one surface via dithiol.
【図４】図３の球を用いることによって、細胞膜中の特
定膜蛋白質と膜面方向の検出方法を示す図。FIG. 4 is a diagram showing a method for detecting a specific membrane protein in a cell membrane and a membrane surface direction by using the sphere of FIG.
【図５】アルミニウム蒸着により、アミノ基とヒドロキ
シル基修飾面を片面づづ形成させ、抗体と蛍光色素をそ
れぞれの面に結合させる方法を示す図。FIG. 5 is a diagram showing a method of forming an amino group- and hydroxyl group-modified surface on each side by aluminum vapor deposition, and bonding an antibody and a fluorescent dye to each surface.
【図６】電気双極子モ−メントに由来する、電場下にお
けるポリスチレン球の配向を示す図。FIG. 6 is a view showing the orientation of polystyrene spheres under an electric field, which is derived from an electric dipole moment.
【図７】抗原抗体反応の伴って変化するポリスチレン球
の応答特性を利用した免疫診断の原理図。FIG. 7 is a principle diagram of immunodiagnosis using the response characteristics of polystyrene spheres that change with an antigen-antibody reaction.
【図８】フォトリトグラフィ−による酸化シリコン微小
担体の製造する方法を示す図。FIG. 8 is a diagram showing a method for producing a silicon oxide microcarrier by photolithography.
【図９】酸化シリコン微小担体の両面に、異なる特性の
蛍光色素を結合する方法を示す図。FIG. 9 is a view showing a method of binding fluorescent dyes having different characteristics to both surfaces of a silicon oxide microcarrier.
【図１０】図９にある微小担体が有する光学、電気特性
のｐＨ依存性を示す図。10 is a diagram showing pH dependence of optical and electrical characteristics of the microcarrier shown in FIG.
【図１１】片面が修飾された球状担体の実例を示す写
真。FIG. 11 is a photograph showing an example of a spherical carrier having one surface modified.
１…球状担体、２…ディスク状担体、３…修飾面
（Ａ）、４…修飾面（Ｂ）、５…修飾面（Ｃ）、６…立
方体状担体、７…修飾領域（Ａ）、８…修飾領域
（Ｂ）、９…棒状担体、１０…基板、１１…未修飾微小
担体、１２…蒸着材料、１３…薄膜、１４…修飾材料、
１５…片面のみ修飾された微小担体、１６…全面修飾さ
れた微小担体、１７…照射、１８…修飾機能損失面、２
０…蛍光色素を含有するポリスチレン球、２１…基板、
２２…金、２３…金薄膜、２４…ジチオ−ル分子層、２
５…スルフヒドリル基と結合する抗体、２６…抗体、３
０…膜蛋白質、３１…リン脂質二重膜、３２…蛍光、４
０…アミン、４１…アミンで修飾されたポリスチレン
球、４２…基板、４３…銀、４４…銀薄膜、４５…アミ
ンと結合する抗体、４６…蒸着されていない表面、４７
…カルボキシル基を有する蛍光色素、４８…酸化銀、４
９…二種類修飾化ポリスチレン球、５０…電荷、５１…
表面電荷、５２…電気双極子モ−メント、６０…電場、
６１…抗原、６２…抗原の電荷、６３…蛍光、７０…電
極、７１…セル、７２…被検体液、７３…変調電位、７
４…光源、７５…光学フィルタ−、７６…光センサ−、
７７…蛍光信号、７８…振幅、７９…特性曲線、８０…
カットオフ周波数、８１…反応後の特性曲線、８２…反
応後に高周波側に移動したカットオフ周波数、９０…シ
リコン基板、９１…酸化シリコン、９２…ポジ型フォト
レジスト、９３…光マスク、９４…照射領域、９５…Ｃ
ＨＦ３ガス、９６…プラズマエッチングで除去された酸
化シリコン、９７…未現像のポジ型フォトレジスト、９
８…酸化シリコン微小担体、９９…エッチングされたシ
リコン基板、１００…基板、１０１…アルミニウム、１
０２…ヒドロキシル基層、１０３…スルフヒドリル基
層、１０４…蛍光色素（Ａ）、１０５…蛍光色素
（Ｂ）、１０６…二種類の蛍光色素で修飾された酸化シ
リコン微小担体、１０７…蛍光色素（Ａ）の蛍光スペク
トル、１０８…蛍光色素（Ｂ）の蛍光スペクトル、１１
０…ｐＨ＜ｐＫ（Ｂ）時の蛍光スペクトル、１１１…ｐ
Ｋ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）時の蛍光スペクトル、１１
２…ｐＫ（Ａ）＜ｐＨ時の蛍光スペクトル、１１３…ｐ
Ｋ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）時の電気双極子モ−メン
ト、１１４…ｐＨ＜ｐＫ（Ｂ）時の電気双極子モ−メン
ト１１５…ｐＫ（Ａ）＜ｐＨ時の電気双極子モ−メン
ト。DESCRIPTION OF SYMBOLS 1 ... Spherical carrier, 2 ... Disc-shaped carrier, 3 ... Modification surface (A), 4 ... Modification surface (B), 5 ... Modification surface (C), 6 ... Cubic carrier, 7 ... Modification area (A), 8 ... Modified region (B), 9 ... Rod-shaped carrier, 10 ... Substrate, 11 ... Unmodified microcarrier, 12 ... Deposition material, 13 ... Thin film, 14 ... Modification material,
15 ... Microcarriers modified on only one side, 16 ... Microcarriers modified on the entire surface, 17 ... Irradiation, 18 ... Modification loss surface, 2
0 ... Polystyrene spheres containing fluorescent dye, 21 ... Substrate,
22 ... Gold, 23 ... Gold thin film, 24 ... Dithiol molecular layer, 2
5 ... Antibody that binds to sulfhydryl group, 26 ... Antibody, 3
0 ... Membrane protein, 31 ... Phospholipid bilayer membrane, 32 ... Fluorescence, 4
0 ... Amine, 41 ... Amine-modified polystyrene spheres, 42 ... Substrate, 43 ... Silver, 44 ... Silver thin film, 45 ... Amine-binding antibody, 46 ... Undeposited surface, 47
... Fluorescent dye having a carboxyl group, 48 ... Silver oxide, 4
9 ... Two types of modified polystyrene spheres, 50 ... Electric charge, 51 ...
Surface charge, 52 ... electric dipole moment, 60 ... electric field,
61 ... Antigen, 62 ... Antigen charge, 63 ... Fluorescence, 70 ... Electrode, 71 ... Cell, 72 ... Analyte fluid, 73 ... Modulation potential, 7
4 ... Light source, 75 ... Optical filter, 76 ... Optical sensor,
77 ... Fluorescence signal, 78 ... Amplitude, 79 ... Characteristic curve, 80 ...
Cutoff frequency, 81 ... Characteristic curve after reaction, 82 ... Cutoff frequency moved to the high frequency side after reaction, 90 ... Silicon substrate, 91 ... Silicon oxide, 92 ... Positive photoresist, 93 ... Photomask, 94 ... Irradiation Area, 95 ... C
HF3 gas, 96 ... Silicon oxide removed by plasma etching, 97 ... Undeveloped positive photoresist, 9
8 ... Silicon oxide microcarrier, 99 ... Etched silicon substrate, 100 ... Substrate, 101 ... Aluminum, 1
02 ... Hydroxyl group layer, 103 ... Sulfhydryl base layer, 104 ... Fluorescent dye (A), 105 ... Fluorescent dye (B), 106 ... Silicon oxide microcarrier modified with two kinds of fluorescent dyes, 107 ... Fluorescent dye (A) Fluorescence spectrum, 108 ... Fluorescence spectrum of fluorescent dye (B), 11
Fluorescence spectrum at 0 ... pH <pK (B), 111 ... p
Fluorescence spectrum when K (B) <pH <pK (A), 11
2 ... pK (A) <pH fluorescence spectrum, 113 ... p
Electric dipole moment when K (B) <pH <pK (A), 114 ... Electric dipole moment when pH <pK (B) 115 ... Electric dipole moment when pK (A) <pH -Ment.
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成８年９月２０日[Submission date] September 20, 1996
【手続補正１】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】図面の簡単な説明[Correction target item name] Brief description of drawings
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【図面の簡単な説明】[Brief description of drawings]
【図１】本発明により製造、使用される不均一的修飾ま
たは異方性形状微小担体の例を示す図。FIG. 1 is a diagram showing an example of a heterogeneous modified or anisotropic shaped microcarrier produced and used according to the present invention.
【図２】蒸着による微小担体の不均一的修飾方法の概念
図。FIG. 2 is a conceptual diagram of a method of nonuniformly modifying a microcarrier by vapor deposition.
【図３】片面のみに金蒸着されたポリスチレン球に、ジ
チオールを介して抗体を結合させる方法を示す図。FIG. 3 is a view showing a method of binding an antibody to polystyrene spheres having gold vapor-deposited on only one surface via dithiol.
【図４】図３の球を用いることによって、細胞膜中の特
定膜蛋白質と膜面方向の検出方法を示す図。FIG. 4 is a diagram showing a method for detecting a specific membrane protein in a cell membrane and a membrane surface direction by using the sphere of FIG.
【図５】アルミニウム蒸着により、アミノ基とヒドロキ
シル基修飾面を片面づづ形成させ、抗体と蛍光色素をそ
れぞれの面に結合させる方法を示す図。FIG. 5 is a diagram showing a method of forming an amino group- and hydroxyl group-modified surface on each side by aluminum vapor deposition, and bonding an antibody and a fluorescent dye to each surface.
【図６】電気双極子モーメントに由来する、電場下にお
けるポリスチレン球の配向を示す図。FIG. 6 is a diagram showing the orientation of polystyrene spheres under an electric field, which is derived from the electric dipole moment.
【図７】抗原抗体反応の伴って変化するポリスチレン球
の応答特性を利用した免疫診断の原理図。FIG. 7 is a principle diagram of immunodiagnosis using the response characteristics of polystyrene spheres that change with an antigen-antibody reaction.
【図８】フォトリトグラフィーによる酸化シリコン微小
担体の製造する方法を示す図。FIG. 8 is a diagram showing a method for producing a silicon oxide microcarrier by photolithography.
【図９】酸化シリコン微小担体の両面に、異なる特性の
蛍光色素を結合する方法を示す図。FIG. 9 is a view showing a method of binding fluorescent dyes having different characteristics to both surfaces of a silicon oxide microcarrier.
【図１０】図９にある微小担体が有する光学、電気特性
のｐＨ依存性を示す図。10 is a diagram showing pH dependence of optical and electrical characteristics of the microcarrier shown in FIG.
【図１１】片面が修飾された球状担体の実例を示す顕微
鏡写真。FIG. 11 is a microscopic view showing an example of a spherical carrier with one surface modified.
Mirror photo.
【符号の説明】 １…球状担体、２…ディスク状担体、３…修飾面
（Ａ）、４…修飾面（Ｂ）、５…修飾面（Ｃ）、６…立
方体状担体、７…修飾領域（Ａ）、８…修飾領域
（Ｂ）、９…棒状担体、１０…基板、１１…未修飾微小
担体、１２…蒸着材料、１３…薄膜、１４…修飾材料、
１５…片面のみ修飾された微小担体、１６…全面修飾さ
れた微小担体、１７…照射、１８…修飾機能損失面、２
０…蛍光色素を含有するポリスチレン球、２１…基板、
２２…金、２３…金薄膜、２４…ジチオール分子層、２
５…スルフヒドリル基と結合する抗体、２６…抗体、３
０…膜蛋白質、３１…リン脂質二重膜、３２…蛍光、４
０…アミン、４１…アミンで修飾されたポリスチレン
球、４２…基板、４３…銀、４４…銀薄膜、４５…アミ
ンと結合する抗体、４６…蒸着されていない表面、４７
…カルボキシル基を有する蛍光色素、４８…酸化銀、４
９…二種類修飾化ポリスチレン球、５０…電荷、５１…
表面電荷、５２…電気双極子モーメント、６０…電場、
６１…抗原、６２…抗原の電荷、６３…蛍光、７０…電
極、７１…セル、７２…被検体液、７３…変調電位、７
４…光源、７５…光学フィルター、７６…光センサー、
７７…蛍光信号、７８…振幅、７９…特性曲線、８０…
カットオフ周波数、８１…反応後の特性曲線、８２…反
応後に高周波側に移動したカットオフ周波数、９０…シ
リコン基板、９１…酸化シリコン、９２…ポジ型フォト
レジスト、９３…光マスク、９４…照射領域、９５…Ｃ
ＨＦ３ガス、９６…プラズマエッチングで除去された酸
化シリコン、９７…未現像のポジ型フォトレジスト、９
８…酸化シリコン微小担体、９９…エッチングされたシ
リコン基板、１００…基板、１０１…アルミニウム、１
０２…ヒドロキシル基層、１０３…スルフヒドリル基
層、１０４…蛍光色素（Ａ）、１０５…蛍光色素
（Ｂ）、１０６…二種類の蛍光色素で修飾された酸化シ
リコン微小担体、１０７…蛍光色素（Ａ）の蛍光スペク
トル、１０８…蛍光色素（Ｂ）の蛍光スペクトル、１１
０…ｐＨ＜ｐＫ（Ｂ）時の蛍光スペクトル、１１１…ｐ
Ｋ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）時の蛍光スペクトル、１１
２…ｐＫ（Ａ）＜ｐＨ時の蛍光スペクトル、１１３…ｐ
Ｋ（Ｂ）＜ｐＨ＜ｐＫ（Ａ）時の電気双極子モーメン
ト、１１４…ｐＨ＜ｐＫ（Ｂ）時の電気双極子モーメン
ト１１５…ｐＫ（Ａ）＜ｐＨ時の電気双極子モーメン
ト。[Explanation of Codes] 1 ... Spherical carrier, 2 ... Disc-shaped carrier, 3 ... Modified surface (A), 4 ... Modified surface (B), 5 ... Modified surface (C), 6 ... Cubic carrier, 7 ... Modified area (A), 8 ... Modified region (B), 9 ... Rod-shaped carrier, 10 ... Substrate, 11 ... Unmodified microcarrier, 12 ... Deposition material, 13 ... Thin film, 14 ... Modification material,
15 ... Microcarriers modified on only one side, 16 ... Microcarriers modified on the entire surface, 17 ... Irradiation, 18 ... Modification loss surface, 2
0 ... Polystyrene spheres containing fluorescent dye, 21 ... Substrate,
22 ... Gold, 23 ... Gold thin film, 24 ... Dithiol molecular layer, 2
5 ... Antibody that binds to sulfhydryl group, 26 ... Antibody, 3
0 ... Membrane protein, 31 ... Phospholipid bilayer membrane, 32 ... Fluorescence, 4
0 ... amine, 41 ... amine-modified polystyrene spheres, 42 ... substrate, 43 ... silver, 44 ... silver thin film, 45 ... amine-binding antibody, 46 ... non-deposited surface, 47
... Fluorescent dye having a carboxyl group, 48 ... Silver oxide, 4
9 ... Two types of modified polystyrene spheres, 50 ... Electric charge, 51 ...
Surface charge, 52 ... electric dipole moment, 60 ... electric field,
61 ... Antigen, 62 ... Antigen charge, 63 ... Fluorescence, 70 ... Electrode, 71 ... Cell, 72 ... Analyte fluid, 73 ... Modulation potential, 7
4 ... Light source, 75 ... Optical filter, 76 ... Optical sensor,
77 ... Fluorescence signal, 78 ... Amplitude, 79 ... Characteristic curve, 80 ...
Cutoff frequency, 81 ... Characteristic curve after reaction, 82 ... Cutoff frequency moved to high frequency side after reaction, 90 ... Silicon substrate, 91 ... Silicon oxide, 92 ... Positive photoresist, 93 ... Photomask, 94 ... Irradiation Area, 95 ... C
HF3 gas, 96 ... Silicon oxide removed by plasma etching, 97 ... Undeveloped positive photoresist, 9
8 ... Silicon oxide microcarrier, 99 ... Etched silicon substrate, 100 ... Substrate, 101 ... Aluminum, 1
02 ... Hydroxyl group layer, 103 ... Sulfhydryl base layer, 104 ... Fluorescent dye (A), 105 ... Fluorescent dye (B), 106 ... Silicon oxide microcarrier modified with two kinds of fluorescent dyes, 107 ... Fluorescent dye (A) Fluorescence spectrum, 108 ... Fluorescence spectrum of fluorescent dye (B), 11
Fluorescence spectrum at 0 ... pH <pK (B), 111 ... p
Fluorescence spectrum when K (B) <pH <pK (A), 11
2 ... pK (A) <pH fluorescence spectrum, 113 ... p
Electric dipole moment when K (B) <pH <pK (A), 114 ... Electric dipole moment when pH <pK (B) 115 ... Electric dipole moment when pK (A) <pH.
Claims (6)
たは異方性形状を持つ微小担体が物理化学的環境に応じ
て配向することを利用した微小領域の計測方法。1. A method for measuring a micro region, which utilizes that a micro carrier having a spherical or anisotropic shape having a region with a specific modification is oriented according to a physicochemical environment.
ン、アビジン、プロティンＡ、プロティンＧ、ペプチ
ド、磁性体、エレクトレットなどの結合性または感受性
物質を用い、さらに不均一的な修飾により、物理化学的
環境に応じて配向する微小担体を利用した請求項１記載
の計測方法。2. Use of a binding or sensitive substance such as an antibody, an antigen, biotin, avidin, protein A, protein G, a peptide, a magnetic substance or an electret for the modification of the microcarrier, and a physical modification by heterogeneous modification. The measuring method according to claim 1, wherein a microcarrier oriented according to a chemical environment is used.
射性同位元素などの標識物質を用い、さらに不均一的な
修飾により配向に応じて標識からの信号が変化する微小
担体を利用した請求項１記載の計測方法。3. A method in which a labeling substance such as a dye, a fluorescent dye, or a radioisotope is used for modifying the microcarrier, and a microcarrier in which a signal from the label changes depending on orientation due to heterogeneous modification is used. Item 1. The measuring method according to item 1.
理的吸着、または化学結合を介し特定な修飾を施された
領域を有する球形または異方性形状を持つ微小担体。4. A microcarrier having a spherical shape or an anisotropic shape, which has a region which is subjected to a specific modification through selective physical adsorption or a chemical bond to a partially formed vapor-deposited film.
機能を部分的に損失させた球形または異方性形状の微小
担体。5. A spherical or anisotropic microcarrier in which the function of modifying the surface of the carrier is partially lost by vapor deposition or irradiation with radiation.
方性形状の微小担体を利用した請求項１記載の計測方
法。6. The measuring method according to claim 1, wherein an anisotropic microcarrier produced by photolithography is used.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12933096A JP3539069B2 (en) | 1996-04-26 | 1996-04-26 | Microcarrier and measurement method using the same |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12933096A JP3539069B2 (en) | 1996-04-26 | 1996-04-26 | Microcarrier and measurement method using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09292395A true JPH09292395A (en) | 1997-11-11 |
| JP3539069B2 JP3539069B2 (en) | 2004-06-14 |
Family
ID=15006940
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12933096A Expired - Fee Related JP3539069B2 (en) | 1996-04-26 | 1996-04-26 | Microcarrier and measurement method using the same |
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| Country | Link |
|---|---|
| JP (1) | JP3539069B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10100819C2 (en) * | 2000-05-12 | 2003-06-12 | Genemaster Lifescience Co | Manufacturing process for microcarriers and test methods with the microcarriers |
| WO2007086189A1 (en) * | 2006-01-30 | 2007-08-02 | Konica Minolta Medical & Graphic, Inc. | Fluorescent labeling substance of nanoparticle or nanorod |
| JP2012198058A (en) * | 2011-03-18 | 2012-10-18 | Hitachi Ltd | Biological molecule measurement system and biological molecule measurement method |
-
1996
- 1996-04-26 JP JP12933096A patent/JP3539069B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10100819C2 (en) * | 2000-05-12 | 2003-06-12 | Genemaster Lifescience Co | Manufacturing process for microcarriers and test methods with the microcarriers |
| WO2007086189A1 (en) * | 2006-01-30 | 2007-08-02 | Konica Minolta Medical & Graphic, Inc. | Fluorescent labeling substance of nanoparticle or nanorod |
| JP2012198058A (en) * | 2011-03-18 | 2012-10-18 | Hitachi Ltd | Biological molecule measurement system and biological molecule measurement method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3539069B2 (en) | 2004-06-14 |
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