Key Laboratory of Luminescent and Real-Time Analytical Chemistry and Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China.
ACS Appl Mater Interfaces. 2015 May 20;7(19):10308-15. doi: 10.1021/acsami.5b00988. Epub 2015 May 5.
An alkaline phosphatase (ALP)-based biosensor can in situ generate an electroactive product by enzymatic hydrolysis of inactive substrates. To obtain a higher signal-to-background ratio, a chemical redox cycling signal-amplified strategy based on the addition of a strong reducing agent has often be applied in the construction of ALP-based biosensors. However, the strong reducing agent not only affects the activity of ALP but also readily reacts with dissolved oxygen, leading to inaccurate results. In this work, a new signal-amplified strategy for a thrombin (TB) aptasensor based on the catalytic oxidation of ALP-generated products, 1-naphthol (NP), using hemin/G-quadruplex DNAzymes was reported. We implemented gold-nanoparticle-decorated zinc oxide nanoflowers (Au-ZnO) as the matrix for immobilizing ALP and TB aptamer (TBA) and then labeled it with hemin to form hemin/G-quadruplex/ALP/Au-ZnO bioconjugates (TBA II bioconjugates). Through a "sandwich" reaction, TBA II bioconjugates were captured on the electrode surface. The amplified signal was carried out in two steps: (i) an ALP-catalyzed inactive substrate, 1-naphthyl phosphate (NPP), in situ produces NP on the surface of the electrode; (ii) on the one hand, NP as a new reactant could be directly electrooxidized and generated an electrochemical signal, but, on the other hand, NP could be oxidized by hemin/G-quadruplex in the presence of H2O2, resulting in amplification of the electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM to 30 nM with a detection limit of 0.37 pM (defined as S/N = 3).
基于碱性磷酸酶 (ALP) 的生物传感器可以通过酶解无活性的底物原位生成电活性产物。为了获得更高的信号背景比，通常在基于 ALP 的生物传感器的构建中应用基于添加强还原剂的化学氧化还原循环信号放大策略。然而，强还原剂不仅会影响 ALP 的活性，而且还会与溶解氧迅速反应，导致结果不准确。在这项工作中，报道了一种基于 ALP 生成产物 1-萘酚 (NP) 的催化氧化的新的凝血酶 (TB) 适体传感器的信号放大策略，使用血红素/G-四链体 DNA 酶。我们采用金纳米颗粒修饰的氧化锌纳米花 (Au-ZnO) 作为固定 ALP 和 TB 适体 (TBA) 的基质，然后用血红素标记形成血红素/G-四链体/ALP/Au-ZnO 生物缀合物 (TBA II 生物缀合物)。通过“三明治”反应，TBA II 生物缀合物被捕获在电极表面上。放大信号分两步进行：(i)ALP 催化无活性底物 1-萘基磷酸酯 (NPP) 在电极表面原位生成 NP；(ii)一方面，NP 可以作为新的反应物直接电氧化并产生电化学信号，但另一方面，NP 可以在 H2O2 的存在下被血红素/G-四链体氧化，导致电化学信号放大。所提出的 TB 适体传感器的线性范围为 1 pM 至 30 nM，检测限为 0.37 pM（定义为 S/N = 3）。
