Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex
Femtomolar Detection of Thrombin in Serum and Cerebrospinal Fluid via Direct Electrocatalysis of Oxygen Reduction by the Covalent G4-Hemin-Aptamer Complex
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DOI：
10.1021/acsami.1c03784
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发表时间：
2021-04-20
影响因子：
9.5
通讯作者：
Ferapontova, Elena E.
中科院分区：
文献类型：
--
作者：
Malecka, Kamila;Ferapontova, Elena E.
凝血酶是一种丝氨酸蛋白酶，在凝血级联反应中起核心作用，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，其低的生理浓度和非特异性结合的其他成分的生理流体复杂的凝血酶的电分析。在这里，飞摩尔水平的凝血酶在血清和人工脑脊液（CSF）检测无指示剂的电化学方法，直接利用O-2还原反应，没有电子转移介质，电催化的共价G4-氯化血红素DNA酶复合物自然自组装后，凝血酶结合到氯化血红素修饰的29聚体DNA适体序列拴到金通过链烷乙氧基化连接器。共吸附的PEG抑制非特异性蛋白质结合，并允许寻求的信号分辨率。所提出的利用G4-氯化血红素DNA酶的“氧化酶”活性的测定不需要使用该DNA酶的传统的基于过氧化物酶活性的测定所必需的任何共反应物，例如H2 O2和氧化还原介体，或溶液脱气，并且允许在充气缓冲液、CSF和1%人血清溶液中快速地、总共30分钟地分析凝血酶。这种开拓性的方法利用氧化酶活性G4-氯化血红素DNAzyme是简单的，灵敏的，和选择性的，并代表了一种新的工具，用于超灵敏的电催化检测的基础上简单而有效的O-2-依赖的DNAzyme标签。
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 O-2 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 O-2-dependent DNAzyme labels.