Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Rebublic of China.
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Rebublic of China; College of Chemistry and Chemical Engineering, Yibin University, Sichuan 644000, China.
Biosens Bioelectron. 2014 May 15;55:313-7. doi: 10.1016/j.bios.2013.12.010. Epub 2013 Dec 11.
Here, an ultrasensitive electrochemiluminescence (ECL) aptasensor using in situ generated proline and polyamidoamine (PAMAM) dendrimers as coreactant for bis(2,2'-bipyridyl)(5-amino-1,10-phenanthroline) ruthenium(II) (Ru) was successfully constructed for detection of thrombin (TB). Firstly, PAMAM combined with PtNPs was used as platform to assemble substantial Ru, prolidase (GPDA) and avidin labeled thrombin aptamer (avidin-TBA) to form PAMAM-PtNPs-Ru-GDPA-TBA bioconjugate. With the double aptamer-based sandwich assay, the bioconjugate was successfully modified on the electrode surface. The proposed aptasensor possessed three attractive advantages: PAMAM, as a tertiary amine substance, not only served as a platform to immobilize the PtNPs for further assembling prolidase (GPDA) and avidin-TBA, but also used as a coreactant of Ru to amplify the ECL signal. Furthermore, putting the coreactant and luminescence reagent together onto the electrode surface could effectively shorten the reaction time, improve the efficiency of electron transfer, and enhance the ECL signal. Lastly, the in situ generated coreactant proline for Ru catalyzed by GPDA could rapidly increase the concentration of proline around the electrode surface, which could also greatly amplify the ECL signal. With the several amplification factors mentioned above, the proposed ECL aptasensor showed a wide linear range from 0.01 pmol L(-1) to 10 nmol L(-1) with the detection limit of 5.0 fmol L(-1). The experimental results also indicated that the aptasensor exhibited excellent selectivity, stability and reproducibility.
这里，构建了一种超灵敏的电化学发光（ECL）适体传感器，使用原位生成的脯氨酸和聚酰胺胺（PAMAM）树状大分子作为共反应物，用于双（2,2'-联吡啶）（5-氨基-1,10-菲啰啉）钌（II）（Ru）的检测。首先，PAMAM 与 PtNPs 结合作为平台，组装大量 Ru、脯氨酸酶（GPDA）和亲和素标记的凝血酶适体（avidin-TBA），形成 PAMAM-PtNPs-Ru-GDPA-TBA 生物缀合物。通过双适体夹心测定法，成功地将生物缀合物修饰在电极表面。所提出的适体传感器具有三个吸引人的优点：PAMAM 作为叔胺物质，不仅用作固定 PtNPs 的平台，进一步组装脯氨酸酶（GPDA）和亲和素-TBA，还用作 Ru 的共反应物，以放大 ECL 信号。此外，将共反应物和发光试剂一起置于电极表面上可以有效地缩短反应时间，提高电子转移效率，并增强 ECL 信号。最后，由 GPDA 催化的 Ru 原位生成的共反应物脯氨酸可以快速增加电极表面周围脯氨酸的浓度，这也可以大大放大 ECL 信号。由于上述几种放大因素，所提出的 ECL 适体传感器在 0.01 pmol L(-1)至 10 nmol L(-1)的宽线性范围内显示出检测限为 5.0 fmol L(-1)。实验结果还表明，该适体传感器表现出优异的选择性、稳定性和重现性。
