JPS6311835A - Measurement for concentration of biomaterial - Google Patents
Measurement for concentration of biomaterialInfo
- Publication number
- JPS6311835A JPS6311835A JP61116626A JP11662686A JPS6311835A JP S6311835 A JPS6311835 A JP S6311835A JP 61116626 A JP61116626 A JP 61116626A JP 11662686 A JP11662686 A JP 11662686A JP S6311835 A JPS6311835 A JP S6311835A
- Authority
- JP
- Japan
- Prior art keywords
- vibrator
- solution
- biosensor
- immobilized
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005259 measurement Methods 0.000 title description 10
- 239000012620 biological material Substances 0.000 title 1
- 239000004816 latex Substances 0.000 claims abstract description 13
- 229920000126 latex Polymers 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000010419 fine particle Substances 0.000 claims description 3
- 239000013078 crystal Substances 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 abstract description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 abstract description 4
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 abstract description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000004471 Glycine Substances 0.000 abstract description 2
- 125000003172 aldehyde group Chemical group 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 abstract 3
- 230000003647 oxidation Effects 0.000 abstract 1
- 238000007254 oxidation reaction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 7
- 230000010355 oscillation Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000010586 diagram Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 101100165177 Caenorhabditis elegans bath-15 gene Proteins 0.000 description 1
- 239000002156 adsorbate Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、生体関連物質の濃度の測定方法に関する。[Detailed description of the invention] [Industrial application field] The present invention relates to a method for measuring the concentration of biologically related substances.
振動子の電極表面に抗体または抗原を固定化した振動子
バイオセンサにおいて、測定溶液中で抗原抗体反応によ
って結合した基質にさらに抗体または抗原を固定化した
ラテックスまたはその他の微粒子を抗原抗体反応によっ
て結合させることによって、振動子バイオセンサの感度
を増幅させた。In a vibrator biosensor in which antibodies or antigens are immobilized on the electrode surface of a vibrator, latex or other fine particles on which antibodies or antigens are further immobilized are bonded to the substrate that has been bound by an antigen-antibody reaction in a measurement solution by an antigen-antibody reaction. By this, the sensitivity of the transducer biosensor was amplified.
振動子バイオセンサによって、抗原抗体反応を利用した
生体関連物質の計測が可能であることが示されている。It has been shown that it is possible to measure biological substances using an antigen-antibody reaction using a transducer biosensor.
振動子バイオセンサは、結合した物質の重■によって応
答が決まるため低濃度の試料や低分子機の物質の計測に
は、十分な感度が得られていなかった。Since the response of a transducer biosensor is determined by the weight of the bound substance, it has not been sensitive enough to measure low-concentration samples or substances in small molecules.
振動子バイオセンサによる測定の際に、抗原または抗体
を固定化した振動子に結合した基質に抗原または抗体を
結合したラテックスまたはその他の微粒子を反応させ、
振動子表面に結合させることによって、振動子表面の重
量を増加させた。During measurement using a transducer biosensor, latex or other fine particles bound with an antigen or antibody are reacted with a substrate bound to a transducer on which the antigen or antibody is immobilized;
By bonding to the vibrator surface, the weight of the vibrator surface was increased.
振動子上に付着した物質の重量が増加するため、振動子
バイオセンサの感度を増幅させることができた。The sensitivity of the transducer biosensor could be amplified due to the increased weight of the substance deposited on the transducer.
（ヒ目ｇＧ ／Ｊｌり定への応用ｌ）
以下、図面に従い本発明のヒ）ＩｇＧ測定への応用につ
いて詳述する。(Application to determination of gG/Jl) Hereinafter, the application of the present invention to h) IgG measurement will be described in detail with reference to the drawings.
第１図＋ａｌは、本発明の原理を示すものであり、１は
水晶振動子電極２の表面に固定化された抗ヒトＩｇＧ抗
体、３は測定基質としてのヒトＩｇＧ 、 ４は感度を
向上させるための抗ヒトＩｇＧ抗体５を固定化したラテ
ックスである。Figure 1+al shows the principle of the present invention, where 1 is an anti-human IgG antibody immobilized on the surface of the crystal resonator electrode 2, 3 is human IgG as a measurement substrate, and 4 is to improve sensitivity. This is latex on which anti-human IgG antibody 5 is immobilized.
水晶振動子バイオセンサの作成は以下の手法に従った。The quartz crystal biosensor was created according to the following method.
まず、パラジウムメッキした水晶振動子の電極表面を０
．５Ｎ Ｎａ０１１中で１時間陽極酸化した。First, the electrode surface of the palladium-plated crystal resonator is
．． Anodized in 5N Na011 for 1 hour.
次に、γ−アミノプロピルトリエトキシシランの１０％
アセトン溶液中で２時間処理した後、５％グルタルアル
デヒド溶液中で３時間処理した。この後、１■／ＩＩＩ
！、の抗ヒ）ＩｇＧ抗体溶液に１時間浸′４して、抗ヒ
トＩｇＧ抗体の固定化を行った。さらに、０．１ Ｍグ
リシン溶液に３０分浸漬して未反応のアルデヒド基の処
理を行った。Next, 10% of γ-aminopropyltriethoxysilane
After being treated in an acetone solution for 2 hours, it was treated in a 5% glutaraldehyde solution for 3 hours. After this, 1■/III
! The anti-human IgG antibody was immobilized by immersing it in an anti-human IgG antibody solution for 1 hour. Furthermore, unreacted aldehyde groups were treated by immersion in a 0.1 M glycine solution for 30 minutes.
本実施例では、フローシステムで測定を行った。In this example, measurements were performed using a flow system.
第２図はフローシステムを示す図である。第２図１８＋
はバイオセンサとセルを示し、第２図１ｂｌはシステム
全体を示す。第２図１８＋において、水晶振動子６の両
面には前述した方法により抗ヒト１ｇＧ抗体が固定され
た電極２が設けられており、それぞれの電極２にはり一
ド７が接続されている。前記水晶振動子６を覆う様にセ
ル８が設けられ、該セルには該セル中に液を循環させる
為にパイプ９が取り付けられている。前記リード７は該
セル８の外部へ引き出されている。引き出された前記リ
ード７は、第２図１８＋に示す様に発振回路ｌＯに接続
されており、また該発振回路１０は、周波数カウンタ１
１に接続されている。前記周波数カウンタ１１には、コ
ンピュータ１２が接続されており前記周波数カウンタ１
１より測定値の収集を行っている。前記水晶振動子６の
外部を取り囲むセル８に設けられたパイプ９の一方は該
セル８中に液を通過させる為のポンプ１３が接続されて
いる。他方のパイプ９は３方パルプ１４の出口に接続さ
れている。選択的に切り換えられる前記３方バルブ１４
の４つの入路のうち１つはそれぞれ恒温槽１５に貯えら
れた蒸留水１６に接続されており、もう１つの入路１７
はＱ、５Ｎ ＮａＣｆｆ１とグリシン塩酸（ｐＨ２，８
）および試料溶液の導入口である。FIG. 2 is a diagram showing the flow system. Figure 2 18+
shows the biosensor and cell, and FIG. 2 1bl shows the entire system. In FIG. 2 18+, electrodes 2 to which anti-human 1gG antibodies are immobilized by the method described above are provided on both surfaces of the crystal resonator 6, and a glue 7 is connected to each electrode 2. A cell 8 is provided so as to cover the crystal oscillator 6, and a pipe 9 is attached to the cell to circulate a liquid through the cell. The lead 7 is led out of the cell 8. The drawn out lead 7 is connected to an oscillation circuit 10 as shown in FIG.
Connected to 1. A computer 12 is connected to the frequency counter 11 and the frequency counter 1
Measured values have been collected since 1. A pump 13 for passing liquid into the cell 8 is connected to one side of a pipe 9 provided in a cell 8 surrounding the outside of the crystal oscillator 6 . The other pipe 9 is connected to the outlet of the three-way pulp 14. said three-way valve 14 which is selectively switched;
One of the four inlets is connected to distilled water 16 stored in a constant temperature bath 15, and the other inlet 17
is Q, 5N NaCff1 and glycine hydrochloric acid (pH 2,8
) and an inlet for the sample solution.
以上の構成において、まずセル中にｐＨ２，８グリシン
−塩ｆ１１溶液を流し、吸着物を除去した１次に、セル
中を蒸留水で置換し、蒸留水を一定速度で流しながら、
発振周波数Ｆ、の測定を行った０次に、ヒ）ＩｇＧｔＳ
液をセル中に流し３０分間反応させた。In the above configuration, first, a pH 2.8 glycine-salt f11 solution is flowed into the cell to remove the adsorbed matter.Then, the inside of the cell is replaced with distilled water, and while the distilled water is flowed at a constant speed,
The 0th order where the oscillation frequency F was measured, h) IgGtS
The solution was poured into the cell and reacted for 30 minutes.
なお、ヒト１ｇＧｉｅｔ［は、３７℃として反応させた
。Note that human 1gGiet [was reacted at 37°C.
この後、０．５Ｎ Ｎａ（ｌを通液して非特異吸゛着物
を除去した。ここで、再び発振周波数Ｆ２を測定した。Thereafter, 0.5N Na (l) was passed through the tube to remove non-specific adsorbents.The oscillation frequency F2 was then measured again.
さらに、抗ヒトＩｇＧ抗体を固定化したラテックスをセ
ル中に流し３０分間反応さセた。再び、０．５ＮＮａＣ
ｌを通液したのち、発振１８波数Ｆ、を測定した。第１
表にＩｇＧに対する応答値（Ｆ＋ Ｆｚ ）とラテ
ックスによって増幅された応答値（Ｆ＋ −Ｆ３）を比
較して示す。表かられかるように、ラテックスによって
感度の増幅が可能であることがわかった。Furthermore, latex immobilized with anti-human IgG antibody was poured into the cell and allowed to react for 30 minutes. Again, 0.5N NaC
After passing through the solution, the oscillation wave number F was measured at 18. 1st
The table shows a comparison of the response value to IgG (F+ Fz) and the response value amplified by latex (F+ -F3). As can be seen from the table, it was found that sensitivity could be amplified by latex.
表
（ヒトＩｇＧ測定への応用２）
以下、図面に従い本発明のヒトＩｇＧ測定への応用につ
いて詳述する。Table (Application 2 to Human IgG Measurement) The application of the present invention to human IgG measurement will be described below in detail with reference to the drawings.
第１図（ｂｌは、本発明の原理を示すものであり、１９
は、水晶振動子電極２の表面に固定化されたプロティン
八、３は測定基質としてのヒトＩＢＧ 、４は感度を向
上させるための抗ヒトＩｇＧ抗体（マウス＋ｇＧ、）
５を固定化したラテックスである。FIG. 1 (bl shows the principle of the present invention, 19
8 is a protein immobilized on the surface of the crystal resonator electrode 2, 3 is human IBG as a measurement substrate, and 4 is an anti-human IgG antibody (mouse+gG) to improve sensitivity.
5 is immobilized in latex.
水晶振動子バイオセンサの作成は、（応用１）で示した
方法と同様に行い、プロティン八を固定化した。測定シ
ステムについても（応用１）と同様のシステムを用いた
。A quartz crystal biosensor was created in the same manner as in (Application 1), and protein 8 was immobilized. As for the measurement system, the same system as in (Application 1) was used.
以上の構成において、まずセル中にＰＨ２，８グリシン
−塩酸緩衝液を流し、吸着物を除去した。次に、セル中
を蒸留水で置換し、蒸留水を一定測度で流しながら、発
振周波数Ｆ１の測定を行っな。In the above configuration, first, a PH2,8 glycine-hydrochloric acid buffer was flowed into the cell to remove adsorbed substances. Next, replace the inside of the cell with distilled water, and measure the oscillation frequency F1 while flowing distilled water at a constant rate.
次に、ヒ）ＩｇＧ？Ｓ液をセル中に流し３０分間反応さ
せた。この後、０．５Ｍ Ｎａｃｌを流し、非特異吸着
物を除去した。セルを蒸留水で置換し、再び発振周波数
Ｆ２を測定した。さらに、抗と）ＩｇＧ抗体（マウスＩ
ｇＧ＋）を固定化したラテックスをセル中に流し３０分
間反応させた。ＰＨ６のリン酸緩衝液を通液し、プロテ
ィン八に直接結合したラテックスを除去したのち、セル
を蒸留水で置換し、発振周波数Ｆ、を測定した。Next, h) IgG? The S solution was poured into the cell and reacted for 30 minutes. After this, 0.5M NaCl was flowed to remove non-specific adsorbates. The cell was replaced with distilled water, and the oscillation frequency F2 was measured again. In addition, anti-)IgG antibodies (mouse I
The latex immobilized with gG+) was poured into the cell and reacted for 30 minutes. After passing a PH6 phosphate buffer solution through the cell to remove latex directly bound to protein 8, the cell was replaced with distilled water, and the oscillation frequency F was measured.
以上で得られたヒ）ＩｇＧに対する反応値ΔＦ ＋−ｔ
（Ｆ＋ Ｆｚ）とラテックスによって増幅された応
答値ΔＦ ＋−ｘ（Ｆ ＋ −Ｆ ｓ）において、ΔＦ
、−３は、ΔＦ ＋−ｔの値に対応して、増幅された値
を示すことがわかった。このことから、抗ヒトＩｇＧ固
定化ラテックスによって感度の向上が可能であることが
わかった。h) Reaction value for IgG obtained above ΔF +-t
(F + Fz) and the response value ΔF + -x (F + -F s) amplified by latex, ΔF
, -3 was found to indicate an amplified value, corresponding to the value of ΔF + -t. From this, it was found that sensitivity can be improved by anti-human IgG immobilized latex.
本発明の振動子バイオセンサの測定方法により、センサ
の感度の増幅が可能になった。The measuring method of the transducer biosensor of the present invention makes it possible to amplify the sensitivity of the sensor.
第１図＋ａ＋及び第１図（ｂｌは、本発明の原理を示す
図であり、第２図は、フローシステムを示す図で、第２
図（ａｌは、バイオセンサとセルの斜視図、第２図（ｂ
ｌはバイオセンサシステム全体を示す概略図である。
以上Figure 1+a+ and Figure 1 (bl are diagrams showing the principle of the present invention, Figure 2 is a diagram showing the flow system,
Figures (al is a perspective view of the biosensor and cell, Figure 2 (b)
1 is a schematic diagram showing the entire biosensor system. that's all
Claims (1)
センサにおいて、抗原抗体反応によって結合した基質に
さらに抗体または抗原を固定化したラテックスまたはそ
の他の微粒子を抗原抗体反応によって結合させることを
特徴とする生体関連物質の濃度測定方法。A transducer biosensor in which antibodies or antigens are immobilized on the surface of the transducer is characterized in that latex or other fine particles on which antibodies or antigens are immobilized are further bonded to the substrate bound by the antigen-antibody reaction by the antigen-antibody reaction. A method for measuring the concentration of biologically related substances.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP86307115A EP0215669A3 (en) | 1985-09-17 | 1986-09-16 | Analytical device and method for analysis of biochemicals, microbes and cells |
| US06/908,371 US4789804A (en) | 1985-09-17 | 1986-09-17 | Analytical device and method utilizing a piezoelectric crystal biosensor |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5165786 | 1986-03-10 | ||
| JP61-51657 | 1986-03-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6311835A true JPS6311835A (en) | 1988-01-19 |
Family
ID=12892948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61116626A Pending JPS6311835A (en) | 1985-09-17 | 1986-05-21 | Measurement for concentration of biomaterial |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6311835A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02249025A (en) * | 1989-03-23 | 1990-10-04 | Matsushita Electric Ind Co Ltd | Signal processor |
| WO2003100386A1 (en) * | 2002-05-28 | 2003-12-04 | National Institute Of Advanced Industrial Science And Technology | Method for measuring trace amount of material by use of quarts resonator |
| JP2007178305A (en) * | 2005-12-28 | 2007-07-12 | Ulvac Japan Ltd | Piezoelectric element and stabilization method of immobilization layer |
| CN108181349A (en) * | 2017-12-21 | 2018-06-19 | 西安交通大学 | The polymer protection material water of historical relic and the test method of salting liquid absorption property |
| JP2020526768A (en) * | 2017-07-11 | 2020-08-31 | アヴィアーナ モレキュラー テクノロジーズ，エルエルシー | Signal amplification in biosensor devices |
-
1986
- 1986-05-21 JP JP61116626A patent/JPS6311835A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02249025A (en) * | 1989-03-23 | 1990-10-04 | Matsushita Electric Ind Co Ltd | Signal processor |
| WO2003100386A1 (en) * | 2002-05-28 | 2003-12-04 | National Institute Of Advanced Industrial Science And Technology | Method for measuring trace amount of material by use of quarts resonator |
| EP1508793A1 (en) * | 2002-05-28 | 2005-02-23 | National Institute of Advanced Industrial Science and Technology | Method for measuring trace amount of material by use of quarts resonator |
| JPWO2003100386A1 (en) * | 2002-05-28 | 2005-09-22 | 独立行政法人産業技術総合研究所 | Method for measuring trace substances using quartz crystal |
| EP1508793A4 (en) * | 2002-05-28 | 2009-09-23 | Nat Inst Of Advanced Ind Scien | Method for measuring trace amount of material by use of quarts resonator |
| JP2007178305A (en) * | 2005-12-28 | 2007-07-12 | Ulvac Japan Ltd | Piezoelectric element and stabilization method of immobilization layer |
| JP2020526768A (en) * | 2017-07-11 | 2020-08-31 | アヴィアーナ モレキュラー テクノロジーズ，エルエルシー | Signal amplification in biosensor devices |
| CN108181349A (en) * | 2017-12-21 | 2018-06-19 | 西安交通大学 | The polymer protection material water of historical relic and the test method of salting liquid absorption property |
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