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 Type	Single-year Grants
Research Field	工業分析化学
Research Institution	Kumamoto 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)
Keywords	Chemiluminescence / 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.