JP3793786B2 - Method for preventing colloidal aggregation - Google Patents
Method for preventing colloidal aggregation Download PDFInfo
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- JP3793786B2 JP3793786B2 JP26194595A JP26194595A JP3793786B2 JP 3793786 B2 JP3793786 B2 JP 3793786B2 JP 26194595 A JP26194595 A JP 26194595A JP 26194595 A JP26194595 A JP 26194595A JP 3793786 B2 JP3793786 B2 JP 3793786B2
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- buffer
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Description
【0001】
【発明の属する技術分野】
本発明は、主に臨床検査で用いられる金属コロイドの凝集を防止する方法に関する。
【0002】
【従来の技術】
金コロイドといった金属コロイドは、臨床検査などで、検査対象物質を標識するために使用されたり、表面増強ラマン散乱の検出系で使用されている。
【0003】
通常、金属コロイドは、その希薄な金属塩溶液から還元することによって製造される(化学的還元法)。還元剤の種類としては、アスコルビン酸,クエン酸,水素化ホウ素ナトリウム,および水素ガスが挙げられる。
【0004】
これら化学的還元法で製造された金属コロイドは、その酸化金属アニオンの由来により、負に帯電した表面を持つ。こういった場合に金属コロイドが安定化するためには、静電的な機構が関与している(”COLLOIDAL GOLD”(ACADEMIC PRESS,INC)を参照されたい)。この静電的な機構が破壊されることによって、金属コロイドどうしが集まり、凝集が起こる。
【0005】
組織染色等の組織科学の分野で使用される金コロイド法がある。そこでは、金コロイドを分散させている溶媒の組成が静電的な機構を破壊し、コロイドを凝集させている。一般に、リン酸塩緩衝液,ホウ酸緩衝液,生理食塩水,EDTA塩緩衝液,酢酸塩緩衝液,グリシン−塩酸緩衝液が金コロイドの溶媒組成に使用されている。
【0006】
また、表面増強ラマン散乱を生起するときにも、金属コロイドが使用される。この金属コロイドを分散させる溶媒は、ラマン活性のない緩衝液組成が用いられる(一例として、リン酸緩衝液)。しかし、これらの緩衝液組成中に金属コロイドを分散させると、先述の金コロイド法と同様に金属コロイドが凝集し、表面増強ラマン散乱が生起しなくなる。
【0007】
したがって、表面増強ラマン散乱を生起するときには、コロイド溶液に安定剤を添加してコロイドの凝集を防止することが一般的に行われている。その安定剤として、天然由来の高分子(牛血清アルブミンや,γ−グロブリン等)や、合成高分子(ポリビニルアルコール,ポリビニルピロリドン等のゲル状物質や、Tween20の様な界面活性剤等)がある。
【0008】
【発明が解決しようとする課題】
上記のように、従来の緩衝液組成ではコロイドを安定に分散させてコロイドの凝集を防止するために、安定剤の添加作業が必須である。よって本発明の目的は、安定剤を必要とせずとも、コロイドの凝集を阻止しようとするものである。
【0009】
【課題を解決するための手段】
上記課題を解決するためには、無機イオンを含まず、かつ有機系である緩衝液を用いることでよいことが判った。
【0010】
【発明の実施の形態】
無機イオンを含まず、かつ有機系である緩衝液の例として、トリスヒドロキシメチルアミノメタン,N−トリスヒドロキシメチル−2−アミノメタンスルホン酸,3−シクロヘキシルアミノプロパンスルホン酸,N−(2−ヒドロキシエチル)ピペラジン−N’−2−エタンスルホン酸の緩衝液が挙げられ、これらを1種の単独で、または2種以上の組合せで使用する。
【0011】
ただし、緩衝液の濃度が20mMを越えると緩衝液そのものに対して表面増強ラマン散乱が生起されてしまい、緩衝液の濃度は高い方が望ましいために、緩衝液の最終濃度は20mMである場合が、コロイドを安定に分散させてコロイドの凝集を防止するために最も好ましい。
【0012】
本発明の優れている点は、コロイドの凝集を防止するだけでなく、この緩衝液を用いることで分散の安定効果が従来の「緩衝液と安定剤」組成と比較して優れている点である。
【0013】
【実施例】
(実施例1) 金コロイドに対する凝集防止効果の確認
25mMリン酸塩緩衝液40mlに対して、安定剤である10%ポリビニルピロリドン5mlを添加した(緩衝液の最終濃度20mM)ものと、安定剤の代わりに蒸留水5mlを添加したものを調製した。それぞれに金コロイド溶液(粒径20nm,OD250=5.0,バイオセル社製)5mlを添加し、450〜700nmでの吸収スペクトルを測定した。結果を図1中(A)に示す。
一方、25mMトリスヒドロキシアミノメタン緩衝液40mlに対して、安定剤である10%ポリビニルピロリドン5mlを添加した(緩衝液の最終濃度20mM)ものと、安定剤の代わりに蒸留水5mlを添加したものを調製した。それぞれに金コロイド溶液(粒径20nm,OD250=5.0,バイオセル社製)5mlを添加し、450〜700nmでの吸収スペクトルを測定した。結果を図1中(B)に示す。
図から、リン酸塩緩衝液では安定剤の有無でスペクトルは非常に変化するが、トリスヒドロキシアミノメタンを緩衝液として用いると、安定剤を添加せずともコロイド溶液の吸収スペクトルは減少しなかった。これはすなわち、金コロイドの凝集が起きていないことを示している。
【0014】
(実施例2) 銀コロイドに対する凝集防止効果の確認
25mMリン酸塩緩衝液40mlに対して、安定剤である10%ポリビニルピロリドン5mlを添加した(緩衝液の最終濃度20mM)ものと、安定剤の代わりに蒸留水5mlを添加したものを調製した。それぞれに銀コロイド溶液(0.1M硝酸銀と0.2M水素化ホウ素ナトリウムとから作成した)5mlを添加し、300〜700nmでの吸収スペクトルを測定した。結果を図2中(A)に示す。
一方、25mMトリスヒドロキシアミノメタン緩衝液40mlに対して、安定剤である10%ポリビニルピロリドン5mlを添加した(緩衝液の最終濃度20mM)ものと、安定剤の代わりに蒸留水5mlを添加したものを調製した。それぞれに銀コロイド溶液(0.1M硝酸銀と0.2M水素化ホウ素ナトリウムとから作成した)5mlを添加し、300〜700nmでの吸収スペクトルを測定した。結果を図2中(B)に示す。
図から、リン酸塩緩衝液では安定剤の有無でスペクトルは非常に変化するが、トリスヒドロキシアミノメタンを緩衝液として用いると、安定剤を添加せずともコロイド溶液の吸収スペクトルは減少しなかった。これはすなわち、銀コロイドの凝集が起きていないことを示している。
【0015】
(実施例3) 金コロイドの凝集防止に対する安定性の確認
25mMN−トリスヒドロキシメチル−2−アミノメタンスルホン酸緩衝液45mlに、金コロイド溶液(粒径20nm,OD250=5.0,バイオセル社製)5mlを添加(緩衝液の最終濃度20mM)し、その520nmにおける吸光度を経時的に測定した。
対照として、25mMリン酸塩緩衝液45mlに金コロイド溶液5mlを添加して、その520nmにおける吸光度も経時的に測定した。
その結果、リン酸塩緩衝液では吸光度が早期に減少し、凝集現象を示すが、N−トリスヒドロキシメチル−2−アミノメタンスルホン酸緩衝液では吸光度の変化は非常に緩やかで、凝集しにくいという結果を得た。結果を図3に示す。
【0016】
(実施例4) 銀コロイドの凝集防止に対する安定性の確認
25mMN−トリスヒドロキシメチル−2−アミノメタンスルホン酸緩衝液45mlに、銀コロイド溶液(0.1M硝酸銀と0.2M水素化ホウ素ナトリウムとから作成した)5mlを添加(緩衝液の最終濃度20mM)し、その490nmにおける吸光度を経時的に測定した。
対照として、25mMリン酸塩緩衝液45mlに銀コロイド溶液5mlを添加して、その490nmにおける吸光度も経時的に測定した。
その結果、リン酸塩緩衝液では吸光度が早期に減少し、凝集現象を示すが、N−トリスヒドロキシメチル−2−アミノメタンスルホン酸緩衝液では吸光度の変化は非常に緩やかで、凝集しにくいという結果を得た。結果を図4に示す。
【0017】
【発明の効果】
詳述のように、本発明を用いると、安定剤の添加をすることなしに、金コロイド法及び表面増強ラマン散乱に用いられる金属コロイドを凝集させることはない。また、分散時の安定効果も優れている。
【0018】
【図面の簡単な説明】
図1中(A)は、リン酸塩緩衝液での金コロイドに対する吸収スペクトルである。
図1中(B)は、トリスヒドロキシアミノメタン緩衝液での金コロイドに対する吸収スペクトルである。
図2中(A)は、リン酸塩緩衝液での銀コロイドに対する吸収スペクトルである。
図2中(B)は、トリスヒドロキシアミノメタン緩衝液での銀コロイドに対する吸収スペクトルである。
図3は、520nmでの金コロイドの吸光度の経時変化である。
図4は、490nmでの銀コロイドの吸光度の経時変化である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for preventing aggregation of metal colloids mainly used in clinical examinations.
[0002]
[Prior art]
Metal colloids such as gold colloids are used for labeling substances to be examined in clinical examinations and the like, and are used in detection systems for surface enhanced Raman scattering.
[0003]
Usually, metal colloids are produced by reduction from their dilute metal salt solution (chemical reduction method). Examples of the reducing agent include ascorbic acid, citric acid, sodium borohydride, and hydrogen gas.
[0004]
The metal colloid produced by these chemical reduction methods has a negatively charged surface due to the origin of the metal oxide anion. In these cases, an electrostatic mechanism is involved in stabilizing the metal colloid (see “COLLOIDAL GOLD” (ACADEMIC PRESS, INC)). When this electrostatic mechanism is destroyed, metal colloids gather and agglomerate.
[0005]
There are colloidal gold methods used in the field of tissue science such as tissue staining. There, the composition of the solvent in which the gold colloid is dispersed destroys the electrostatic mechanism and agglomerates the colloid. In general, phosphate buffer solution, borate buffer solution, physiological saline, EDTA salt buffer solution, acetate buffer solution and glycine-hydrochloric acid buffer solution are used for the solvent composition of gold colloid.
[0006]
Metal colloids are also used when generating surface enhanced Raman scattering. As the solvent for dispersing the metal colloid, a buffer composition having no Raman activity is used (for example, a phosphate buffer). However, when the metal colloid is dispersed in these buffer compositions, the metal colloid aggregates as in the gold colloid method described above, and surface enhanced Raman scattering does not occur.
[0007]
Therefore, when surface-enhanced Raman scattering occurs, a stabilizer is generally added to the colloid solution to prevent colloid aggregation. As the stabilizer, there are naturally-derived polymers (bovine serum albumin, γ-globulin, etc.), synthetic polymers (gel substances such as polyvinyl alcohol and polyvinylpyrrolidone, and surfactants such as Tween 20). .
[0008]
[Problems to be solved by the invention]
As described above, in the conventional buffer solution composition, in order to stably disperse the colloid and prevent the colloid from aggregating, an operation of adding a stabilizer is essential. The object of the present invention is therefore to prevent colloidal aggregation without the need for stabilizers.
[0009]
[Means for Solving the Problems]
In order to solve the above problem is not free of inorganic ions, and buffer it was found that good at are use the organic system.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Free of inorganic ions, and examples of the buffer is an organic, trishydroxymethylaminomethane, N- tris (hydroxymethyl) methyl-2-amino-methanesulfonic acid, 3-cyclohexylamino-propanesulfonic acid, N- (2-hydroxy Ethyl) piperazine-N′-2-ethanesulfonic acid buffers, which may be used singly or in combination of two or more.
[0011]
However, if for concentration of buffer will be occurring surface-enhanced Raman scattering with respect to the buffer itself exceeds 20mM, the concentration of the buffer is higher is desirable, the final concentration of the buffer is 20mM Is most preferable in order to stably disperse the colloid and prevent aggregation of the colloid.
[0012]
The superior point of the present invention is not only the prevention of colloidal aggregation, but also the use of this buffer solution is superior in dispersion stability compared to the conventional “buffer and stabilizer” composition. is there.
[0013]
【Example】
(Example 1) Confirmation of anti-aggregation effect on colloidal gold To 40 ml of 25 mM phosphate buffer, 5 ml of 10% polyvinylpyrrolidone as a stabilizer was added (final concentration of buffer 20 mM), and Instead, 5 ml of distilled water was added. 5 ml of colloidal gold solution (particle size 20 nm, OD 250 = 5.0, manufactured by Biocell) was added to each, and the absorption spectrum at 450 to 700 nm was measured. The results are shown in FIG.
On the other hand, 40 ml of 25 mM Trishydroxyaminomethane buffer was added with 5 ml of 10% polyvinylpyrrolidone as a stabilizer (final buffer concentration 20 mM), and 5 ml of distilled water instead of the stabilizer. Prepared. 5 ml of colloidal gold solution (particle size 20 nm, OD 250 = 5.0, manufactured by Biocell) was added to each, and the absorption spectrum at 450 to 700 nm was measured. The results are shown in FIG.
From the figure, the spectrum of the phosphate buffer varies greatly depending on the presence or absence of a stabilizer, but when trishydroxyaminomethane was used as the buffer, the absorption spectrum of the colloidal solution did not decrease without the addition of a stabilizer. . This indicates that no aggregation of colloidal gold has occurred.
[0014]
(Example 2) Confirmation of anti-aggregation effect on silver colloid To 40 ml of 25 mM phosphate buffer, 5 ml of 10% polyvinylpyrrolidone as a stabilizer was added (final concentration of buffer 20 mM), and Instead, 5 ml of distilled water was added. 5 ml of a silver colloid solution (made from 0.1 M silver nitrate and 0.2 M sodium borohydride) was added to each, and the absorption spectrum at 300 to 700 nm was measured. The results are shown in FIG.
On the other hand, 40 ml of 25 mM Trishydroxyaminomethane buffer was added with 5 ml of 10% polyvinylpyrrolidone as a stabilizer (final buffer concentration 20 mM), and 5 ml of distilled water instead of the stabilizer. Prepared. 5 ml of a silver colloid solution (made from 0.1 M silver nitrate and 0.2 M sodium borohydride) was added to each, and the absorption spectrum at 300 to 700 nm was measured. The results are shown in FIG.
From the figure, the spectrum of the phosphate buffer varies greatly depending on the presence or absence of a stabilizer, but when trishydroxyaminomethane was used as the buffer, the absorption spectrum of the colloidal solution did not decrease without the addition of a stabilizer. . This indicates that no aggregation of silver colloid has occurred.
[0015]
(Example 3) Confirmation of stability against aggregation of colloidal gold In 45 ml of 25 mM N-trishydroxymethyl-2-aminomethanesulfonic acid buffer, colloidal gold solution (particle size 20 nm, OD 250 = 5.0, manufactured by Biocell) ) 5 ml was added (final concentration of buffer 20 mM), and the absorbance at 520 nm was measured over time.
As a control, 5 ml of colloidal gold solution was added to 45 ml of 25 mM phosphate buffer, and the absorbance at 520 nm was also measured over time.
As a result, in the phosphate buffer solution, the absorbance decreases early and shows an aggregation phenomenon, but in the N-trishydroxymethyl-2-aminomethanesulfonic acid buffer solution, the change in absorbance is very gradual and hardly aggregates. The result was obtained. The results are shown in FIG.
[0016]
(Example 4) Confirmation of stability against aggregation of silver colloid In 45 ml of 25 mM N-trishydroxymethyl-2-aminomethanesulfonic acid buffer, a silver colloid solution (from 0.1 M silver nitrate and 0.2 M sodium borohydride) was added. 5 ml (prepared) was added (the final concentration of the buffer was 20 mM), and the absorbance at 490 nm was measured over time.
As a control, 5 ml of silver colloid solution was added to 45 ml of 25 mM phosphate buffer, and the absorbance at 490 nm was also measured over time.
As a result, in the phosphate buffer solution, the absorbance decreases early and shows an aggregation phenomenon, but in the N-trishydroxymethyl-2-aminomethanesulfonic acid buffer solution, the change in absorbance is very gradual and hardly aggregates. The result was obtained. The results are shown in FIG.
[0017]
【The invention's effect】
As detailed, the present invention does not agglomerate metal colloids used in gold colloid methods and surface enhanced Raman scattering without the addition of stabilizers. Moreover, the stabilizing effect at the time of dispersion | distribution is also excellent.
[0018]
[Brief description of the drawings]
In FIG. 1, (A) is an absorption spectrum for colloidal gold in a phosphate buffer.
In FIG. 1, (B) is an absorption spectrum for gold colloid in a trishydroxyaminomethane buffer.
In FIG. 2, (A) is an absorption spectrum for silver colloid in a phosphate buffer.
In FIG. 2, (B) is an absorption spectrum for silver colloid in a trishydroxyaminomethane buffer.
FIG. 3 shows the time course of the absorbance of colloidal gold at 520 nm.
FIG. 4 shows the time course of the absorbance of the colloidal silver at 490 nm.
Claims (2)
無機イオンを含まず、かつ有機系であるトリスヒドロキシメチルアミノメタン,N−トリスヒドロキシメチル−2−アミノメタンスルホン酸,3−シクロヘキシルアミノプロパンスルホン酸,N−(2−ヒドロキシエチル)ピペラジン−N’−2−エタンスルホン酸のうち、1種または2種以上の組合せからなる緩衝液を用いることを特徴とする、金属コロイドの凝集を防止する方法。A method for preventing aggregation of metal colloids,
Trishydroxymethylaminomethane, N-trishydroxymethyl-2-aminomethanesulfonic acid, 3-cyclohexylaminopropanesulfonic acid, N- (2-hydroxyethyl) piperazine-N ′ which does not contain inorganic ions and is organic among 2-ethanesulfonic acid, one or of two or more thereof, characterized in that there use a buffer, a method of preventing aggregation of metal colloid.
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JP26194595A JP3793786B2 (en) | 1995-09-04 | 1995-09-04 | Method for preventing colloidal aggregation |
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JP26194595A JP3793786B2 (en) | 1995-09-04 | 1995-09-04 | Method for preventing colloidal aggregation |
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JP3793786B2 true JP3793786B2 (en) | 2006-07-05 |
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US7198957B2 (en) | 2001-03-09 | 2007-04-03 | Takao Fukuoka | Method for analyzing substance |
JP5364310B2 (en) * | 2008-07-14 | 2013-12-11 | アルフレッサファーマ株式会社 | Method for stabilizing microparticles to which reactive substances are bound, and reagent containing the microparticles |
WO2013147308A1 (en) | 2012-03-30 | 2013-10-03 | 積水メディカル株式会社 | Immunochromatographic test strip and detection method using immunochromatography for detecting target in red blood cell-containing sample |
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