Department of Chemistry, University of Kurdistan, 66177-15175, Sanandaj, Iran.
Biosens Bioelectron. 2014 Feb 15;52:20-8. doi: 10.1016/j.bios.2013.08.012. Epub 2013 Aug 20.
We have developed a sensitive electrochemical immunosensor for the detection of prostate-specific antigen (PSA), based on covalently immobilizing of anti-PSA and redox mediator (thionine) onto gold nanoparticles-incorporated polyamidoamine dendrimer (AuNPs-PAMAM) and multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite (MWCNTs/IL/Chit) as the support platform. The MWCNTs/IL/Chit nanocomposite and synthesized AuNPs were characterized using SEM and TEM microscopy techniques. Greatly amplified immunoassay was established by sandwiching the antigen between anti-PSA immobilized on the MWCNTs/IL/Chit/AuNPs-PAMAM interface and anti-PSA labeled with horseradish peroxidase (HRP-labeled anti-PSA) as secondary antibody. Phtaloyl chloride (Ph) was used as linking agent for the subsequent immobilization of AuNPs-PAMAM onto platform and anti-PSA antibody and thionine onto AuNPs-PAMAM dendrimer. The increased electrocatalytic reduction of H2O2 by HRP was monitored by differential pulse voltammetry technique. Under optimized condition the calibration curve for PSA concentration was linear up to 80 ng ml(-1) with detection limit (signal-to-noise ratio of 3) of 1 pg ml(-1). AuNPs-PAMAM dendrimer as platform not only increased the amount of thionine and PSA antibody but also the electron transfer process accelerated by encapsulated AuNPs. Moreover, the proposed PSA immunosensor exhibited excellent stability and reproducibility. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. In addition, impedance technique was used as simple, rapid, low cost label free analytical method for PSA measurement with detection limit of 0.5 ng ml(-1) at concentration range up to 25 ng ml(-1). The results indicate that the present protocol is quite promising in developing other electrochemical immunosensors.
我们开发了一种基于金纳米粒子-聚酰胺胺树枝状大分子(AuNPs-PAMAM)和多壁碳纳米管/离子液体/壳聚糖纳米复合材料(MWCNTs/IL/Chit)作为支撑平台的电化学免疫传感器,用于检测前列腺特异性抗原(PSA)。MWCNTs/IL/Chit 纳米复合材料和合成的 AuNPs 采用 SEM 和 TEM 显微镜技术进行了表征。通过夹心免疫分析法,将抗原夹在固定在 MWCNTs/IL/Chit/AuNPs-PAMAM 界面上的抗 PSA 和作为二次抗体的辣根过氧化物酶标记的抗 PSA(HRP-labeled anti-PSA)之间,建立了极大放大的免疫测定。用邻苯二甲酰氯(Ph)作为连接剂,将 AuNPs-PAMAM 接枝到平台上,并将抗 PSA 抗体和硫堇接枝到 AuNPs-PAMAM 树枝状大分子上。通过差分脉冲伏安法监测 HRP 对 H2O2 的电化学催化还原。在优化条件下,PSA 浓度的校准曲线在 80ngml(-1) 以下呈线性,检测限(信噪比为 3)为 1pgml(-1)。AuNPs-PAMAM 树枝状大分子作为平台,不仅增加了硫堇和 PSA 抗体的数量,而且还加速了包裹的 AuNPs 的电子传递过程。此外,所提出的 PSA 免疫传感器表现出优异的稳定性和重现性。通过与标准 ELISA 检测的比较,证明了该传感器能够在人血清样品中准确检测 PSA。此外,阻抗技术作为一种简单、快速、低成本的无标记分析方法,用于 PSA 的测量,检测限为 0.5ngml(-1),浓度范围高达 25ngml(-1)。结果表明,该方案在开发其他电化学免疫传感器方面具有很大的应用前景。