Bioassay of bile acids using an enzyme-linked DNA aptamer.
Bioassay of bile acids using an enzyme-linked DNA aptamer.
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DOI：
10.1039/b004580m
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
T. Kato;K. Yano;K. Ikebukuro;I. Karube
中科院分区：
文献类型：
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作者：
T. Kato;K. Yano;K. Ikebukuro;I. Karube
通过采用与胆汁酸结合的碱性磷酸酶连接的 DNA 寡聚物，开发了一种检测胆汁酸的新分析方法。通过体外选择方法确定的长度为 40 个核苷酸的 5'-生物素标记的 DNA 寡聚物通过亲和素-生物素键与碱性磷酸酶连接，并应用于酶免疫测定形式。将样品溶液与胆酸固定琼脂糖基质的小等分试样一起孵育，在进行测定之前，碱性磷酸酶连接的 DNA 寡聚物已结合在琼脂糖基质上。使用碱性磷酸酶的荧光底物检测从胆酸固定的琼脂糖基质中解离的碱性磷酸酶连接的 DNA 寡聚物的量，表明样品中胆汁酸的量。结果表明，直接与报告酶连接的DNA适体可用作免疫测定形式的检测配体。胆酸的线性校准范围为 0.1 至 5 mmol l-1，检测限为 10 mumol l-1。 5 mmol l-1 胆酸时，%RSD 为 7。
A new analytical method for the detection of bile acids has been developed by adopting an alkaline phosphatase-linked DNA oligomer that binds to bile acids. A 5'-biotin-labeled DNA oligomer with a 40-nucleotide length that is defined by the in vitro selection method was connected with alkaline phosphatase through an avidin-biotin linkage and applied to an enzyme immunoassay format. Sample solutions were incubated with small aliquots for a cholic acid-immobilized agarose matrix, on which the alkaline phosphatase-linked DNA oligomer had been bound prior to carrying out the assay. The amount of the alkaline phosphatase-linked DNA oligomer dissociated from the cholic acid-immobilized agarose matrix, which was detected using a fluorogenic substrate for alkaline phosphatase, indicated the amount of bile acids in the samples. The results suggest that the DNA aptamer directly linked with the reporter enzyme is applicable as a detector ligand for the immunoassay format. A linear calibration range was obtained for cholic acid between 0.1 to 5 mmol l-1 with a limit of detection of 10 mumol l-1. The %RSD was 7 at 5 mmol l-1 of cholic acid.