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Determination of Chemiluminescent Labeling Compounds by Photographic Method

Research Project

Project/Area Number 63550568
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 工業分析化学
Research InstitutionKumamoto Institute of Technology

Principal Investigator

UENO Keihei  Kumamoto Inst. Tech. Professor, 工学部, 教授 (60037653)

Co-Investigator(Kenkyū-buntansha) SHIGA Masanobu  Dojindo Laboratories Ltd., 研究部, 研究員
YOSHIDA Isao  Kumamoto Inst. Tech. Assoc. Prof., 工学部, 助教授 (30158430)
SAGARA Fumio  Kumamoto Inst. Tech. Assoc. Prof., 工学部, 助教授
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1988: ¥1,200,000 (Direct Cost: ¥1,200,000)
KeywordsChemiluminescence / Polaroid film / Xray film / luminol / fluorophore / oxalate ester / Phenyl 10-methylacrydinium-9-carboxylate / tetraphenylporphine-Fe complex / Phenylー10ーMethylacridiniumー9ーcarboxylate, / tetraphenylporphineーFe complex. / 超高感度インスタントフィルム / フェナントロリン / ルミノール / Nーロチルアクリジニウムエステル / ポーフィリン鉄 / DNAプローブ
Research Abstract

The research was undertaken to detect ultratrace amount of protein or DNA by photographic detection of light from the protein or DNA which was labeled with chemiluminescent compounds. The advantage of photographic method over photon counting method is that the former gives two-dimensional information after plate electrophoresis of the analytes. The 10^<-14> - 10^<-18> mole level of labeling compounds was detected by our method.
1. Chemiluminescence from phenyl 10-methylacridinium-9-carboxylate (PMAC) - H_2O_2 system. This substrate luminesces by the reaction with H_2O_2 at pH 7 - 12 in the presence of cationic micelle (lambda_<max> 470 nm). For example, 20 mul of 4.86 X 10^-9 M PMAC was placed in a microtiter plate, to which was added with 30 mul of 0.03 M NaOH, 0.186% H_2O_2, 0.1 M CEDAB mixed solution. Emitted light was recorded on a Polaroid Type 612 film (ISO 20,000). A spot corresponding to 9 X 10^<-14> mole was detected by this method.
2. Chemiluminescence from oxalate ester - fluorophor - H_2O_2 system. In this system, fluorophor can be selected fairly widely, but the reaction has to be carried out in a media containing organic solvents. For example, 20 mul of 4.8 X 10^<-9> M pyrene MIBK/acetone (2:3) (containing 0.028 % H_2O_2 and buffered to pH 6) WAS placed on a membrane filter, to which added with 20 mul of 1.0 X 10^<-3> M bis(trichlorophenyl)- oxalate MIBK/acetone solution. Emitted light was recorded on a X ray film (Fuji AIF RX). A spot corresponding to 9 X 10^<-14> mole was detected by this method.
3. Chemiluminescence from Luminol - iron complex -H_2O_2 system. In this system, iron complex can be used as a labeling compound, and 2 X 10^<-18> mole of tetraphenylporphine iron(III) complex (TPP-Fe) was detected by this method. lambda-Phage DNA was labeled with TPP-Fe, and the matching DNA at 10 ng level could be detected on a nitrocellulose membrance filter by photographic detection with a Polaroid type 612 film.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (4 results)

All Other

All Publications (4 results)

  • [Publications] K.UENO,F.SAGRA.,I.YOSHIDA,H.ENAMI,S.ETOH,H.MIYAZAKI,A.SONODA and M.SAITO: "Detection of Chemiluminescent Labeling Compounds by Photographic Method.Preliminary Investigation on 1,10-Phenanthroline-Hydrogen Peroxide and Luminol-Hydrogen Peroxide Systems" Analytical Sciences. VOL.4. 477-480 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.UENO, F.SAGARA, I.YOSHIDA, H.ENAMI, S.ETOH, H.MIYAZAKI, A.SONODA and M.SAITO: "Detection of Chemiluminescent Labeling Compounds by Photographic Method. Preliminary Investigation on 1, 10-Phenanthroline-Hydrogen Peroxide and Luminol-Hydrogen Peroxide Systems" Analytical Sciences. VOL.4. 477-480 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Ueno,F.Sagara,I.Yoshida,H.Enami,S.Etoh,H.Miyazaki,A.Sonoda.and M.Saito: "Detection of Chemiluminescent Labeling Compounds by Photographic Method. Preliminary Investigation on 1,10ーPhenanthrolineーHydrogen Peroxide and LuminolーHydrogen Peroxide Systems" Analytical Sciences. Vol.4. 477-480 (1988)

    • Related Report
      1989 Annual Research Report
  • [Publications] Keihei Ueno,;Fumio Sagara,;Isao Yoshida,;Hiroko Enami,;Shigeki Etoh,;Horoshige Miyazaki: Analytical Sciences. 4. 477-480 (1988)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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