Centi Sonia, Tombelli Sara, Minunni Maria, Mascini Marco
Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy.
Anal Chem. 2007 Feb 15;79(4):1466-73. doi: 10.1021/ac061879p.
The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.
DNA凝血酶适配体已得到广泛研究，并且这种适配体与不同转导原理的结合已证明适配体作为生物受体在生物分析检测中的广泛适用性。这项工作的目标是设计一种基于适配体的夹心检测法，用于在复杂基质中对凝血酶进行电化学检测，该检测法通过适配体功能化磁珠进行简单的目标捕获步骤。首先通过表面等离子体共振优化适配体固定和蛋白质结合的条件，然后将其转移到在丝网印刷电极上进行的基于电化学的检测中。然后将该检测法应用于缓冲液、加标血清和血浆中凝血酶的分析，发现具有高灵敏度和特异性。此外，通过其前体凝血酶原的转化在血浆中原位生成凝血酶，并在不同时间跟踪凝血酶的形成。电化学检测法检测到的浓度与模拟凝血酶随时间形成的模拟软件（血栓图）一致。所提出的工作表明，适配体的高特异性以及磁珠的使用是在复杂基质中进行基于适配体分析的关键特征，为实际应用于诊断或医学研究开辟了可能性。
