Department of Analytical Chemistry, Faculty of Chemistry, University Computense of Madrid, Ciudad Universitaria, 28040 Madrid, Spain.
Biosens Bioelectron. 2010 Oct 15;26(2):517-22. doi: 10.1016/j.bios.2010.07.060. Epub 2010 Jul 22.
A disposable electrochemical immunosensor using screen-printed carbon electrodes (SPCEs) and protein A-functionalized magnetic beads (MBs) was developed for the determination of testosterone. Anti-testosterone was immobilized onto MBs and a direct competitive immunoassay involving testosterone labeled with peroxidase (HRP) was performed. The resulting conjugate was trapped on the SPCE with a small magnet. Testosterone determination was carried out by amperometry at -0.2V upon H2O2 additions using hydroquinone (HQ) as the redox mediator. The experimental variables involved in the immunosensor response to testosterone were evaluated. Under the optimized conditions, a calibration plot for testosterone was obtained with a linear range between 5.0×10(-3) and 50 ng/mL (r=0.995). The detection limit was 1.7 pg/mL and the EC50 was 0.25±0.04 ng/mL. These characteristics are notably better than those achieved with other reported immunosensors. Furthermore, anti-testosterone/MBs conjugates were shown to be stable for at least 25 days. A good selectivity was also found against other steroid hormones. The usefulness of the immunosensor was demonstrated by analyzing human serum spiked with 1 and 10 ng/mL testosterone.
一种基于丝网印刷碳电极（SPCE）和蛋白 A 功能化磁珠（MBs）的一次性电化学免疫传感器被开发出来，用于检测睾酮。将抗睾酮固定在 MBs 上，并进行涉及用过氧化物酶（HRP）标记的睾酮的直接竞争性免疫分析。所得缀合物用小磁铁捕获在 SPCE 上。通过添加 H2O2 并用对苯二酚（HQ）作为氧化还原介体在 -0.2V 进行安培法测定睾酮。评估了与睾酮免疫传感器响应相关的实验变量。在优化条件下，获得了睾酮的校准曲线，线性范围为 5.0×10(-3) 至 50ng/mL（r=0.995）。检测限为 1.7pg/mL，EC50 为 0.25±0.04ng/mL。这些特性明显优于其他报道的免疫传感器。此外，抗睾酮/MBs 缀合物在至少 25 天内显示出稳定。还发现对其他甾体激素具有良好的选择性。通过分析添加了 1 和 10ng/mL 睾酮的人血清，证明了免疫传感器的有用性。
