Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 34134, South Korea.
Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 34134, South Korea; Division of Life Science, Korea Basic Science Institute, Daejeon 34133, South Korea.
Biosens Bioelectron. 2017 May 15;91:637-643. doi: 10.1016/j.bios.2017.01.021. Epub 2017 Jan 12.
A novel highly sensitive dendrimer coupled impedimetric immunosensor was developed for the label-free and reagent-free detection of cardiac troponin I (TnI) in serum samples. The immunosensor probe was fabricated by covalently attaching carboxylic acid-functionalized third generation (G3) poly (amidoamine) (PAMAM) dendrimer (Den) on the 3, 3', 5, 5'-tetramethyl benzidine (TMB) modified 6-mercaptohexanoic acid (MHA) self-assembled monolayer (SAM) on a gold (Au) electrode. Monoclonal anti-TnI antibody was then covalently immobilized on the Den and TMB attached MHA SAM modified surface. TMB was used as an internal surface redox couple for generating signal which also allowed to avoid the use of an external one (i.e. ferricyanide couple) in solution during the impedance measurement for monitoring the antibody-antigen binding. On the other hand, Den was used as a signal enhancer by immobilizing more anti-body on the immunosensor probe. The immunosensor probe was characterized using X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) techniques. The TnI detection in diluted serum was based on the measurement of charge transfer resistance (R) of the electron transfer process of the surface-attached TMB before and after immunobinding. Under the optimized condition, the proposed immunosensor could detect human TnI in diluted serum samples as low as 11.7 fM with a wide linear dynamic range, good stability, and excellent specificity. The validity of the proposed method was tested in various TnI spiked human undiluted serum samples and was compared with the enzyme-linked immunosorbent assay (ELISA). The results suggested that the proposed immunosensor could be a useful tool for practical applications in clinical diagnosis.
一种新型的高灵敏度树枝状聚合物耦合阻抗免疫传感器被开发出来，用于无标记和无试剂检测血清样本中的心肌肌钙蛋白 I（TnI）。免疫传感器探针通过将羧酸功能化的第三代（G3）聚（酰胺-胺）（PAMAM）树枝状聚合物（Den）共价连接到 3,3',5,5'-四甲基联苯胺（TMB）修饰的 6-巯基己酸（MHA）自组装单层（SAM）上制备而成，该自组装单层位于金（Au）电极上。然后，将单克隆抗 TnI 抗体共价固定在 Den 和 TMB 连接的 MHA SAM 修饰表面上。TMB 被用作内部表面氧化还原偶联物来产生信号，这也允许在阻抗测量中避免使用溶液中的外部偶联物（即铁氰化物偶联物）来监测抗体-抗原结合。另一方面，Den 被用作信号增强剂，通过将更多的抗体固定在免疫传感器探针上。使用 X 射线光电子能谱（XPS）、石英晶体微天平（QCM）、循环伏安法（CV）和电化学阻抗谱（EIS）技术对免疫传感器探针进行了表征。在稀释的血清中，TnI 的检测是基于免疫结合前后表面附着的 TMB 的电子转移过程的电荷转移电阻（R）的测量。在优化条件下，该免疫传感器能够以 11.7 fM 的低浓度检测稀释血清中的人 TnI，具有较宽的线性动态范围、良好的稳定性和优异的特异性。该方法的有效性在各种 TnI 掺杂的人未稀释血清样本中进行了测试，并与酶联免疫吸附测定（ELISA）进行了比较。结果表明，该免疫传感器可以作为临床诊断中实际应用的有用工具。
