Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea.
Department of Periodontics, Seoul St Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Biosens Bioelectron. 2019 Feb 1;126:122-128. doi: 10.1016/j.bios.2018.10.040. Epub 2018 Oct 23.
This research aims to develop biosensors which could diagnose periodontal diseases in early phases and predict the illness stage of patients, in order to give them adequate treatment timely. Human odontogenic ameloblast-associated protein (ODAM) is considered to be a potential biomarker for periodontal diseases, based on high correlation between the level of ODAM in gingival crevicular fluid (GCF) and the degree of periodontitis. Many aptamers, including a cognate pair of aptamers which can bind to the different sites of ODAM, were successfully screened in a very stringent condition employing saliva as a counter target through the graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX). For the characterization of the aptamer candidates, GO-based fluorescence resonance energy transfer (GO-FRET) and surface plasmon resonance (SPR) assays were conducted. The sandwich-type binding of a cognate pair of aptamers to ODAM was additionally confirmed by employing circular dichroism (CD) and magnetic beads-based fluorescence imaging methods. The resulting cognate pair of aptamers, OD64 and OD35, were found to have their dissociation constant (Kd), 47.71 nM and 51.36 nM, respectively. The minimum detectable concentrations of a sandwich-type SPR biosensor were found to be 0.24 nM and 1.63 nM, respectively, for both buffered and saliva samples. Finally, using this cognate pair of aptamers, a sandwich-type lateral flow strip biosensor was successfully realized. This research shows the potential for implementation of a cognate pair of aptamers on point-of-care biosensors which enables simple, rapid, and non-invasive saliva-based diagnosis of periodontal-related diseases that can overcome current diagnostic methods and improve health care system.
本研究旨在开发能够在早期阶段诊断牙周病并预测患者疾病阶段的生物传感器，以便及时为他们提供适当的治疗。人类牙源性成釉细胞相关蛋白（ODAM）被认为是牙周病的潜在生物标志物，因为龈沟液（GCF）中 ODAM 的水平与牙周炎的程度高度相关。通过在非常严格的条件下使用唾液作为对照靶标，在基于氧化石墨烯的配体指数富集系统进化（GO-SELEX）中成功筛选了许多适体，包括可以结合 ODAM 不同位点的同源对适体。为了对适体候选物进行表征，进行了基于氧化石墨烯的荧光共振能量转移（GO-FRET）和表面等离子体共振（SPR）测定。通过使用圆二色性（CD）和基于磁性珠的荧光成像方法，进一步证实了同源对适体与 ODAM 的三明治型结合。所得到的同源对适体 OD64 和 OD35 的解离常数（Kd）分别为 47.71 nM 和 51.36 nM。夹心型 SPR 生物传感器的最低检测浓度分别为 0.24 nM 和 1.63 nM，对于缓冲液和唾液样本均如此。最后，使用该同源对适体，成功实现了三明治型侧向流动条生物传感器。本研究表明，将同源对适体应用于即时护理生物传感器具有潜力，这使得基于简单、快速、非侵入性唾液的牙周相关疾病的诊断成为可能，从而克服当前的诊断方法并改善医疗保健系统。
