富勒醇-FITC磷光标记固体基质室温磷光法测定痕量碱性磷酸酶  

Solid Substrate RoomTemperature Phosphorimetry for the Determination of Trace Alkaline Phosphatase Using Fullerol-fluorescein Isothiocyanate as Labelling Reagent

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作  者:林少琴[1] 

机构地区:[1]福建教育学院理科研修部,福建福州350001

出  处:《漳州师范学院学报(自然科学版)》2011年第1期63-69,共7页Journal of ZhangZhou Teachers College(Natural Science)

基  金:福建省自然科学基金项目(2009J01017);福建省教育厅科技项目(JA10277)

摘  要:开发了新的富勒醇、异硫氰酸荧光素和N,N-二甲基苯胺(F-ol-(FITC)n-DMA)标记试剂.基于F-ol的活性-OH与FITC游离的-COOH反应生成含有多个FITC分子的复合物(F-ol-(FITC)n-DMA),增加WGA-AP-WGA-F-ol FITC-DMA(WGA和AP分别为麦胚凝集素与碱性磷酸酶)生物靶的发光分子数而进一步提高SSRTP(固体基质室温磷光法)的灵敏度.用F-ol-(FITC)n-DMA标记WGA方法的检出限为0.15 ag spot-1(F-ol)与0.097 ag spot-1((FITC),比F-ol-DMA、FITC-DEA单发光分子标记WGA方法的检出限(0.20 ag spot-1(F-ol),0.22 ag spot-1(FITC)低.用于血清试样中AP含量的测定结果与酶联免疫法相吻合.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.

关 键 词:碱性磷酸酶 富勒醇-异硫氰酸荧光素 麦胚凝集素 亲和吸附固体基质室温磷光法 

分 类 号:O629.8[理学—有机化学]

 

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