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题名: Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay
作者: Guo, Limin; Zhao, Qiang
刊名: MICROCHIMICA ACTA
出版日期: 2016-12
卷号: 183, 期号:12, 页码:3229-3235
关键词: Aptasensor ; Fluorescence ; Enzyme label ; Microplate assay ; Microtiter plate ; Fluorogenic enzyme substrate ; Serine protease ; DNA probe ; Affinity binding ; Dual functional probe ; Displacement ; Indirect analysis
DOI: 10.1007/s00604-016-1978-1
部门归属: 环境化学与生态毒理学国家重点实验室
英文摘要: The authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum.
收录类别: SCI
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内容类型: 期刊论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35834
Appears in Collections:环境化学与生态毒理学国家重点实验室_期刊论文

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