Jarczewska Marta, Kékedy-Nagy László, Nielsen Jesper S, Campos Rui, Kjems Jørgen, Malinowska Elżbieta, Ferapontova Elena E
Interdisciplinary Nanoscience Center (iNANO) and Center for DNA Nanotechnology (CDNA), Aarhus University, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark.
Analyst. 2015 Jun 7;140(11):3794-802. doi: 10.1039/c4an02354d.
Protein biomarkers of cancer allow a dramatic improvement in cancer diagnostics as compared to the traditional histological characterisation of tumours by enabling a non-invasive analysis of cancer development and treatment. Here, an electrochemical label-free assay for urokinase plasminogen activator (uPA), a universal biomarker of several cancers, has been developed based on the recently selected uPA-specific fluorinated RNA aptamer, tethered to a gold electrode via a phosphorothioated dA tag, and soluble redox indicators. The binding properties of the uPA-aptamer couple and interference from the non-specific adsorption of bovine serum albumin (BSA) were modulated by the electrode surface charge. A nM uPA electroanalysis at positively charged surfaces, complicated by the competitive adsorption of BSA, was tuned to the pM uPA analysis at negative surface charges of the electrode, being improved in the presence of negatively charged BSA. The aptamer affinity for uPA displayed via the binding/dissociation constant relationship correspondingly increased, ca. three orders of magnitude, from 0.441 to 367. Under optimal conditions, the aptasensor allowed 10(-12)-10(-9) M uPA analysis, also in serum, being practically useful for clinical applications. The proposed strategy for optimization of the electrochemical protein sensing is of particular importance for the assessment and optimization of in vivo protein ligand binding by surface-tethered aptamers.
与通过肿瘤传统组织学特征进行诊断相比，癌症蛋白质生物标志物能够对癌症发展和治疗进行非侵入性分析，从而显著改善癌症诊断。在此，基于最近筛选出的uPA特异性氟化RNA适配体，开发了一种用于尿激酶型纤溶酶原激活剂（uPA，多种癌症的通用生物标志物）的电化学免标记检测方法，该适配体通过硫代磷酸化dA标签连接到金电极上，并结合可溶性氧化还原指示剂。电极表面电荷调节了uPA-适配体对的结合特性以及牛血清白蛋白（BSA）非特异性吸附的干扰。在带正电表面进行的纳摩尔级uPA电分析因BSA的竞争性吸附而变得复杂，通过调节电极表面电荷为负，可实现皮摩尔级uPA分析，且在存在带负电的BSA时得到改善。适配体对uPA的亲和力通过结合/解离常数关系相应增加，约三个数量级，从0.441增加到367。在最佳条件下，该适配体传感器能够对血清中的uPA进行10⁻¹² - 10⁻⁹ M的分析，在临床应用中具有实际用途。所提出的优化电化学蛋白质传感的策略对于评估和优化表面连接适配体在体内的蛋白质配体结合尤为重要。
