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 |
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Authors: | Jia-Ming Liu Xiao-Mei Huang Zhen-Bo Liu Fei-Ming Li Zhi-Ming Li Lian-Ying Li |
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Affiliation: | a Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000, PR China b The Third Hospital of Xiamen, Xiamen 361100, PR China c Department of Biochemistry, Fujian Education College, Fuzhou 350001, China d Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou 363000, PR China |
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Abstract: | 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. |
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Keywords: | Alkaline phosphatase Fullerol-fluorescein isothiocyanate Wheat germ agglutinin Affinity adsorption solid substrate room temperature phosphorimetry |
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