Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.
A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.
Liu
Jia-Ming
JM
Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000, PR China. zzsyliujiaming@163.com
Huang
Xiao-Mei
XM
Liu
Zhen-Bo
ZB
Lin
Shao-Qin
SQ
Li
Fei-Ming
FM
Gao
Fei
F
Li
Zhi-Ming
ZM
Zeng
Li-Qing
LQ
Li
Lian-Ying
LY
Ouyang
Ying
Y
D000469:Alkaline Phosphatase / Q000097:blood*; D000814:Aniline Compounds / Q000737:chemistry*; D004194:Disease / Q000209:etiology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents; D017879:Isothiocyanates / Q000737:chemistry; D008163:Luminescent Measurements; D013050:Spectrometry, Fluorescence; D014909:Wheat Germ Agglutinins / Q000737:chemistry
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D014909:Wheat Germ Agglutinins; C037152:isothiocyanic acid; D000469:Alkaline Phosphatase