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基于被截断适配体引起的氧化石墨烯增强荧光偏振变化的荧光法测定脂多糖

Fluorometric determination of lipopolysaccharides via changes of the graphene oxide-enhanced fluorescence polarization caused by truncated aptamers.

机构信息

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.

College of Food Engineering, Anhui Science and Technology University, Fengyang, 233100, China.

出版信息

Mikrochim Acta. 2019 Feb 15;186(3):173. doi: 10.1007/s00604-019-3261-8.

Abstract

A broad-spectrum ssDNA aptamer containing 80 nucleotides (LA80) and capable of binding to four different sources of lipopolysaccharides (LPSs) was truncated. Two strategies are used to produce truncated aptamers of different length. The results show that LA27, a 27-nt aptamer, retained broad-spectrum capability and has a higher affinity (K = 46.2 ± 9.5 nM). A graphene oxide based fluorescence polarization assay (excitation/emission wavelengths: 485/520 nm) was worked out using FAM-labeled LA27. It can detect LPSs from Salmonella entericaserotype typhimurium, Pseudomonas aeruginosa 10 and Escherichia coli 055:B5 with enhanced performance (4.8 to 29-fold improvements) compared to LA80. The assay can be performed within 30 min, and the detection limits are 38.7, 88.0 and 154 ng·mL, respectively. Graphical abstract Schematic presentation of the assay: A shorter aptamer, with higher affinity than its original aptamer, was obtained by truncated strategies. This core aptamer lead to release easily and enhance the sensivity of the GO-based fluorescence polarization assay.

摘要

一种含有 80 个核苷酸的广谱 ssDNA 适体(LA80),能够结合四种不同来源的脂多糖(LPS),被截断。使用两种策略来产生不同长度的截断适体。结果表明,27 个核苷酸的 LA27 保留了广谱能力,并且具有更高的亲和力(K=46.2±9.5 nM)。使用 FAM 标记的 LA27 开发了基于氧化石墨烯的荧光偏振测定法(激发/发射波长:485/520nm)。与 LA80 相比,它可以检测到来自沙门氏菌血清型伤寒、铜绿假单胞菌 10 和大肠杆菌 055:B5 的 LPSs,性能得到增强(4.8 到 29 倍的提高)。该测定法可在 30 分钟内完成,检测限分别为 38.7、88.0 和 154ng·mL。

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