Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex,ACS Applied Materials & Interfaces

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Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2021-04-20 , DOI: 10.1021/acsami.1c03784
Kamila Malecka _{1,

2} , Elena E Ferapontova ₁

Affiliation

Thrombin, a serine protease playing a central role in the coagulation cascade reactions and a potent neurotoxin produced by injured brain endothelial cells, is a recognized cardiac biomarker and a critical biomarker for Alzheimer’s disease development. Both in vivo and in vitro, its low physiological concentrations and nonspecific binding of other components of physiological fluids complicate electroanalysis of thrombin. Here, femtomolar levels of thrombin in serum and an artificial cerebrospinal fluid (CSF) were detected by the indicator-free electrochemical methodology exploiting the O₂ reduction reaction directly, with no electron transfer mediators, electrocatalyzed by the covalent G4-hemin DNAzyme complex naturally self-assembling upon thrombin binding to the hemin-modified 29-mer DNA aptamer sequence tethered to gold via an alkanethiol linker. Coadsorbed PEG inhibited nonspecific protein binding and allowed the sought signal resolution. The proposed assay exploiting the “oxidase” activity of G4-hemin DNAzyme does not require any coreactants necessary for the traditional peroxidase activity-based assays with this DNAzyme, such as H₂O₂ and redox mediators, or solution deaeration and allows fast, overall 30 min analysis of thrombin in aerated buffer, CSF, and 1% human serum solutions. This pioneer approach exploiting the oxidase activity G4-hemin DNAzyme is simple, sensitive, and selective and represents a new tool for ultrasensitive electrocatalytic assays based on simple and efficient O₂-dependent DNAzyme labels.

中文翻译：

通过共价 G4-血红素-适体复合物直接电催化氧还原对血清和脑脊液中凝血酶的飞摩尔检测

凝血酶是一种在凝血级联反应中起核心作用的丝氨酸蛋白酶，也是一种由受损脑内皮细胞产生的强效神经毒素，是公认的心脏生物标志物和阿尔茨海默病发展的关键生物标志物。在体内和体外，它的低生理浓度和与生理液体其他成分的非特异性结合使凝血酶的电分析变得复杂。_{在这里，通过利用 O 2}的无指示剂电化学方法检测血清和人工脑脊液 (CSF) 中凝血酶的飞摩尔水平直接还原反应，没有电子转移介质，由共价 G4-血红素 DNAzyme 复合物在凝血酶结合到通过烷硫醇接头拴在金上的血红素修饰的 29-mer DNA 适体序列时自然自组装进行电催化。共吸附的 PEG 抑制非特异性蛋白质结合并允许寻求信号分辨率。所提出的利用 G4-血红素 DNA 酶的“氧化酶”活性的测定法不需要使用这种 DNA 酶进行传统的基于过氧化物酶活性的测定法所必需的任何共反应物，例如 H ₂ O ₂和氧化还原介质，或溶液脱气，并允许对充气缓冲液、CSF 和 1% 人血清溶液中的凝血酶进行快速、整体的 30 分钟分析。这种利用氧化酶活性 G4-血红素 DNAzyme 的开创性方法简单、灵敏且具有选择性，代表了一种基于简单有效的 O ₂依赖性 DNAzyme 标记的超灵敏电催化测定的新工具。

更新日期：2021-04-20

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