Although surface plasmon resonance (SPR) technique and aptamer technology shows great potential in analytical and biological chemistry, direct capture and analysis of small molecules using SPR remains tough. Detection sensitivity of aptasensor and recognition ability of aptamer is limited, because direct immobilization of aptamer causes large steric hindrance and strand entanglement. Herein, we chose a typical small molecule-tetracycline (Mw. 444.4 g/mol) as a model, and combined aptamer technology, DNA nanostructure, and commercial Biacore T200 SPR instrument to develop a straightforward format SPR aptasensor. Anti-tetracycline aptamer (Apt76) was fabricated on the top of a tetrahedron nanostructure to provide a better accessibility to tetracycline than the single-stranded Apt76 (ss-Apt76), and thus to improve sensitivity of the SPR aptasensor. The aptasensor was then validated in real world application for tetracycline screening in multiple honey samples, achieving good recovery rates of 80.20–114.3%, intuitive sensorgrams indicating the binding kinetic properties, and high specificity towards tetracycline. LOD of the tetrahedron-based SPR aptasensor was obtained using the real honey sample and calculated to be 0.0069 μg/kg, which was 10-fold range lower than that of the ss-Apt76-based aptasensor. The proof-of-concept demonstrated that aptamers of small molecules can be oriented immobilized on the SPR surface in a uniform nanoscale distance in both lateral and vertical direction, so as to achieve better conformational folding and better accessibility to small molecules. The concept is promising to be a universal and powerful tool for other ligand immobilization and SPR studies for both real world detection and molecular interaction.

尽管表面等离振子共振（SPR）技术和适体技术在分析和生物化学中显示出巨大潜力，但是使用SPR直接捕获和分析小分子仍然很困难。适体传感器的检测灵敏度和适体的识别能力受到限制，因为直接固定适体会导致较大的空间位阻和链缠结。在这里，我们选择典型的小分子四环素（分子量444.4 g / mol）作为模型，并结合适体技术，DNA纳米结构和商用Biacore T200 SPR仪器来开发一种简单的格式SPR适体传感器。在四面体纳米结构的顶部制造了抗四环素适体（Apt76），以提供比单链Apt76（ss-Apt76）更好的四环素可及性，从而提高了SPR适体传感器的灵敏度。aptasensor随后在多个蜂蜜样品中用于四环素筛选的实际应用中得到验证，实现了80.20–114.3％的良好回收率，直观的传感图表明了结合动力学特性，并且对四环素具有很高的特异性。使用真实的蜂蜜样品获得了基于四面体的SPR适体传感器的LOD，其LOD计算为0.0069μg/ kg，这比基于ss-Apt76的适体传感器的LOD低10倍。概念验证表明，小分子的适体可以在横向和垂直方向上以均匀的纳米级距离定向固定在SPR表面上，从而实现更好的构象折叠和更好的小分子可及性。