Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan.
Biosens Bioelectron. 2010 Apr 15;25(8):1922-7. doi: 10.1016/j.bios.2010.01.005. Epub 2010 Jan 18.
We unveil a novel, label-free, colorimetric assay--using fibrinogen (Fib) and gold nanoparticles (Au NPs)--for the highly selective and sensitive detection of thrombin. Addition of fibrinogen to a solution of Au NPs (average diameter: 56 nm) led to ready conjugation, forming Fib-Au NPs through electrostatic and hydrophobic interactions. Introduction of thrombin (a serine protease) into the Fib-Au NPs solutions in the presence of excess fibrinogen induced the formation of insoluble fibrillar fibrin-Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. After centrifugation, the absorbance at 532 nm of the supernatants decreased upon increasing the concentration of thrombin. This Fib-Au NP probe provides high sensitivity [limit of detection (LOD): 0.04 pM] for thrombin, with remarkable selectivity over other proteins and proteases. The range of linearity for the absorbance against the thrombin concentration was 0.1-10 pM (R(2)=0.96). This approach provides an LOD for thrombin that is lower than those obtainable using other nanomaterial- and aptamer-based detection methods. We validated the utility of this Fib-Au NP probe through separate analyses of thrombin and Factor Xa at picomolar levels in plasma samples--without the need for sample pretreatment. This technique appears to have practical potential in the diagnosis of diseases associated with coagulation abnormalities and cancers (e.g., pulmonary metastasis).
我们揭示了一种新颖的、无标记的比色分析方法——使用纤维蛋白原（Fib）和金纳米粒子（Au NPs）——用于高度选择性和灵敏地检测凝血酶。将纤维蛋白原添加到 Au NPs（平均直径：56nm）溶液中会导致其易于结合，通过静电和疏水相互作用形成 Fib-Au NPs。在存在过量纤维蛋白原的情况下，将凝血酶（一种丝氨酸蛋白酶）引入 Fib-Au NPs 溶液中，会通过未结合和结合的纤维蛋白原的聚合诱导不溶性纤维状纤维蛋白原-Au NPs 聚集物的形成。离心后，上清液在 532nm 处的吸光度随着凝血酶浓度的增加而降低。这种 Fib-Au NP 探针对凝血酶具有高灵敏度[检测限（LOD）：0.04pM]，对其他蛋白质和蛋白酶具有显著的选择性。吸光度与凝血酶浓度之间的线性范围为 0.1-10pM（R(2)=0.96）。这种方法提供的凝血酶 LOD 低于其他基于纳米材料和适体的检测方法。我们通过在血浆样品中分别分析皮摩尔级别的凝血酶和因子 Xa 验证了这种 Fib-Au NP 探针的实用性——无需样品预处理。这种技术似乎在与凝血异常和癌症（例如肺转移）相关的疾病的诊断中具有实际潜力。
