Center for Advanced Analytical Science, School of Chemistry and Chemical Engineering , Guangzhou University , Guangzhou 510006 , P. R. China.
School of Civil Engineering , Guangzhou University , Guangzhou 510006 , P. R. China.
Anal Chem. 2020 Feb 18;92(4):3470-3476. doi: 10.1021/acs.analchem.9b05647. Epub 2020 Jan 29.
As a serine protease, thrombin is a pivotal component in coagulation cascade and has been frequently screened as an informative biomarker for the diagnosis of coagulation disorder-related diseases. Herein, a "signal-on" electrochemical biosensor is described for the highly sensitive and selective detection of thrombin activity, by exploiting a thrombin-specific substrate peptide (Tb peptide) as the recognition element and reversible addition-fragmentation chain transfer (RAFT) polymerization for signal amplification. Specifically, the carboxyl-group-free Tb peptides are self-assembled onto gold electrode surface via the N-terminal cysteine residue and are used for the specific recognition of thrombin molecules. After the proteolytic cleavage of the Tb peptides, the carboxyl-group-containing RAFT agents (4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, CPAD) are tethered to the free carboxyl termini of the truncated peptide fragments via the carboxylate-zirconium-carboxylate chemistry. The subsequent RAFT polymerization leads to the grafting of a polymer chain from each proteolytically cleaved site, enabling the recruitment of a large number of electroactive ferrocene (Fc) tags to the electrode surface when ferrocenylmethyl methacrylate (FcMMA) is used as the monomer. Under optimal conditions, the detection limit of the described thrombin biosensor is as low as 2.7 μU mL (∼0.062 pM), with a linear response over the range of 10-250 μU mL ( = 0.997). Results also indicate that the biosensor is highly selective and applicable to the detection of thrombin activity in complex serum samples and the screening of thrombin inhibitors. The described biosensor is low-cost and relatively easy in preparation and thus shows great promise for the highly sensitive and selective detection of thrombin activity.
作为一种丝氨酸蛋白酶，凝血酶是凝血级联反应的关键组成部分，经常被筛选为凝血障碍相关疾病的诊断的信息生物标志物。在此，通过利用凝血酶特异性底物肽（Tb 肽）作为识别元件和可逆加成-断裂链转移（RAFT）聚合进行信号放大，描述了一种“信号开启”电化学生物传感器，用于高灵敏和选择性检测凝血酶活性。具体而言，无羧基的 Tb 肽通过 N 端半胱氨酸残基自组装到金电极表面，并用于凝血酶分子的特异性识别。在 Tb 肽的蛋白水解切割后，通过羧酸-锆酸-羧酸化学将含羧基的 RAFT 试剂（4-氰基-4-（苯甲酰硫基）戊酸，CPAD）连接到截断肽片段的游离羧基末端。随后的 RAFT 聚合导致从每个蛋白水解切割位点接枝聚合物链，当使用 2-（全氟丁基）乙基甲基丙烯酸酯（FEMA）作为单体时，可将大量电活性的二茂铁（Fc）标记物募集到电极表面。在最佳条件下，所描述的凝血酶生物传感器的检测限低至 2.7 μU mL（∼0.062 pM），线性响应范围为 10-250 μU mL（ = 0.997）。结果还表明，该生物传感器具有高度选择性，可用于复杂血清样品中凝血酶活性的检测和凝血酶抑制剂的筛选。所描述的生物传感器成本低，制备相对简单，因此在高灵敏和选择性检测凝血酶活性方面具有广阔的应用前景。
