Ding Yanjun, Wang Hua, Shen Guoli, Yu Ruqin
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China.
Anal Bioanal Chem. 2005 Aug;382(7):1491-9. doi: 10.1007/s00216-005-3350-x. Epub 2005 Jul 9.
A highly sensitive electrochemical immunoassay for Toxoplasma gondii-specific IgG (Tg-IgG) in human serum has been developed that is based on an enzyme-catalyzed amplification due to the formation of an insoluble precipitate on the surface of a quartz crystal microbalance (QCM). T. gondii antigen (TgAg) was immobilized on the surface of a gold electrode in order to bind Tg-IgG, and this was followed by the addition of anti-Tg-IgG horseradish peroxidase conjugate (anti-Tg-IgG-HRP). Subsequent exposure to 3,3'-diaminobenzidine (DAB) led to the enzymatically-catalyzed amplified deposition of the oxidation products on the QCM surface in the presence of H2O2. The transduction methods electrochemical Faradaic impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to assay the resistance to electron transfer at the conductive support upon accumulation of the insoluble products. The precipitation process was monitored in real time by QCM. The assay conditions, including the concentration of immobilized TgAg and the dosage of anti-Tg-IgG-HRP conjugate, were optimized. It was found that the amount of precipitate that accumulated on the conductive QCM surface was determined by the concentration of the target analyte Tg-IgG and the time permitted for biocatalyzed precipitation. The technique was shown to give a linear electron transfer resistance response (as measured by EIS) for Tg-IgG dilutions ranging between 1:8000 and 1:200, and a detection limit of 1:9600 dilution.
已开发出一种用于检测人血清中弓形虫特异性IgG（Tg-IgG）的高灵敏度电化学免疫分析方法，该方法基于石英晶体微天平（QCM）表面形成不溶性沉淀而导致的酶催化扩增。将弓形虫抗原（TgAg）固定在金电极表面以结合Tg-IgG，随后加入抗Tg-IgG辣根过氧化物酶偶联物（抗Tg-IgG-HRP）。随后在存在H2O2的情况下，暴露于3,3'-二氨基联苯胺（DAB）会导致氧化产物在QCM表面发生酶催化的扩增沉积。采用电化学法拉第阻抗谱（EIS）和循环伏安法（CV）等转导方法来测定不溶性产物积累时导电载体上的电子转移电阻。通过QCM实时监测沉淀过程。对包括固定化TgAg的浓度和抗Tg-IgG-HRP偶联物的用量在内的分析条件进行了优化。结果发现，在导电QCM表面积累的沉淀量取决于目标分析物Tg-IgG的浓度和生物催化沉淀允许的时间。该技术对Tg-IgG稀释倍数在1:8000至1:200之间的样品给出线性电子转移电阻响应（通过EIS测量），检测限为1:9600稀释倍数。
