喹诺酮广谱特异性核酸适配体筛选和多残留传感器构建

Quinolones are a class of widely-used antibiotics. The quinolone residue problem has been a crucial issue in the field of food and environmental safety. The development of a rapid, on-site method for multi-quinolone detection is urgently needed. Immunoassays have been the most prevalent methods for rapid detection of quinolones. However, their application is limited by difficulties in isolation of broad-spectrum antibody, high-cost production and batch-to-batch variations. Nucleic acid-based aptamers have recently become an ideal alternative for antibodies with many advantages such as rapid isolation with tunable specificity and affinity, easy chemical modification for sensor development, high chemical stability, and low production cost. We here propose an aptamer-based method for rapid visible detection of quinolones in food and water samples. The proposed project contains three specific aims: First, development of a parallel-serial aptamer isolation strategy for isolation of a full-spectrum aptamer recognizing the core-structure of quinolones. The isolated aptamer can bind with all quinolone antibiotics with high affinity. Second, characterization of the binding affinity and cross-reactivity of the isolated aptamer. Third, development of a label-free, signal-on assay based on Cy7 displacement for rapid, sensitive and visualized detection of all quinolones. Importantly, the methods developed in this project can also be applied to rapidly generate sensors for broad-spectrum detection of other small molecule targets.

喹诺酮是应用广泛的一类抗生素，其残留问题是食品和环境安全领域的重要议题，发展高效的喹诺酮多残留快速检测方法十分必要。免疫分析法是目前最常用的喹诺酮多残留快速检测法，广谱抗体筛选困难、昂贵且批次差异大，限制了免疫分析法的应用。核酸适配体更易筛选、修饰改造、功能化、稳定、经济，是抗体的理想代替品。本项目围绕喹诺酮广谱性适配体筛选和快速多残留检测方法展开。首先，基于喹诺酮的空间构象差异建立超广谱核酸适配体筛选方法，分别通过并联筛选与串联筛选，获得识别喹诺酮骨架结构的广谱特异性核酸适配体；其次，表征研究适配体的靶标亲和力和特异性；再次，以Cy7作为信号因子，基于三臂交联体结构，构建信号增强、非标记型、可视化、快速、灵敏的检测方法，实现对喹诺酮的多残留检测。所构建的方法有望为其他小分子的广谱核酸适配体筛选和多残留检测提供借鉴。

广谱特异性识别探针是构建药物多残留快速检测传感器的前提，而这类探针分子的筛选一直以来是研究的难点。在本项目的资助下，我们开展了针对小分子药物的广谱特异性核酸适配体筛选研究工作。我们提出了可控的“并联-串联”筛选方法，首先通过平行筛选选择性地富集适配体文库，随后串行筛选以富集与指定目标分子家族特异性结合的适配体。为了证明该方法的可行性，我们将其用于筛选卡西酮广谱核酸适配体，所得适配体对12种不同结构的卡西酮类药物亲和力为纳摩尔级，对11种结构相似的干扰化合物无响应。我们进一步将该方法应用于喹诺酮类抗生素核酸适配体筛选，最终获得5条具有高亲和力、交叉反应性和高特异性的核酸适配体，可同时识别24种喹诺酮类抗生素，亲和力为0.097-5.15 μM，对于其他类抗生素无响应。选择其中Apt.fq02适配体构建基于荧光共振能量转移生物传感器，对牛奶、河水和土壤样品中环丙沙星、帕珠沙星、氟甲喹、恶喹酸、萘啶酸、恩诺沙星、氧氟沙星、诺氟沙星等喹诺酮类抗生素的检测限低至43-95nM。综上所述，我们提出“并联-串联”策略解决了小分子药物的广谱特异性核酸适配体体外筛选的难题，分别获得了1条特异性识别12种卡西酮类药物和5条特异性识别24种喹诺酮类抗生素的核酸适配体，并构建了可用于复杂样品检测的可视化和荧光生物传感器。本项目所构建的适配体筛选和分析方法有望拓展用于其他小分子家族检测，在医疗诊断、食品和环境检测中具有广泛的应用前景。