†State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, P. R. China.
‡School of Life and Science, Yangtze University, Jingzhou 434025, P. R. China.
ACS Appl Mater Interfaces. 2015 Jun 3;7(21):11215-23. doi: 10.1021/acsami.5b01180. Epub 2015 May 19.
Aptamers are short single-stranded DNA or RNA oligonucleotides and can be selected from synthetic combinatorial libraries in vitro. They have a high binding affinity and specificity for their targets. Agarose gels, nitrocellulose membranes, and adsorptive microplates are often used as carriers to immobilize targets in the SELEX (systematic evolution of ligands by exponential enrichment) process, but the subsequent separation step is tedious and time-consuming. Therefore, we used magnetic nanoparticles (MNPs) as carriers to immobilize the target, hepatitis B surface antigen (HBsAg), which is convenient for fast magnetic separation. In this study, we first selected DNA aptamers against HBsAg by immobilizing HBsAg on the surface of carboxylated MNPs. The ssDNA library of each selection round was prepared by asymmetric PCR amplification for the next selection round. To obtain aptamer sequences, the final selected products were purified by gel electrophoresis, then cloned, and sequenced. DNA aptamers that specifically bind to HBsAg were successfully obtained after 13 selection rounds. The selected aptamers were used to construct a chemiluminescence aptasensor based on magnetic separation and immunoassay to detect HBsAg from pure protein or actual serum samples. There was a linear relationship between HBsAg concentration and chemiluminescent intensity in the range of 1-200 ng/mL. The aptasensor worked well even in the presence of interfering substances and was highly specific in the detection of HBsAg in serum samples, with a detection limit 0.1 ng/mL lower than the 0.5 ng/mL limit of an ELISA in use at the hospital. This aptasensor can contribute to better detection of hepatitis B virus infection.
适体是短的单链 DNA 或 RNA 寡核苷酸，可在体外从合成组合文库中筛选得到。它们对其靶标具有高的结合亲和力和特异性。琼脂糖凝胶、硝酸纤维素膜和吸附微板通常用作载体，将靶标固定在 SELEX（配体系统进化的指数富集）过程中，但随后的分离步骤繁琐且耗时。因此，我们使用磁性纳米颗粒（MNPs）作为载体来固定靶标，乙型肝炎表面抗原（HBsAg），这便于快速磁分离。在这项研究中，我们首先通过将 HBsAg 固定在羧基化 MNPs 表面来筛选针对 HBsAg 的 DNA 适体。每个选择轮的 ssDNA 文库通过不对称 PCR 扩增进行下一轮选择的准备。为了获得适体序列，最后选择的产物通过凝胶电泳纯化，然后克隆和测序。经过 13 轮选择，成功获得了特异性结合 HBsAg 的适体。所选适体用于构建基于磁分离和免疫测定的化学发光适体传感器，以检测来自纯蛋白或实际血清样本的 HBsAg。在 1-200ng/mL 的范围内，HBsAg 浓度与化学发光强度之间存在线性关系。该适体传感器在存在干扰物质的情况下仍能很好地工作，并且在检测血清样本中的 HBsAg 时具有高度特异性，检测限比医院使用的 ELISA 低 0.1ng/mL。该适体传感器有助于更好地检测乙型肝炎病毒感染。
