A novel electrochemiluminescence aptasensor based on in situ generated proline and matrix polyamidoamine dendrimers as coreactants for signal amplication

• A sandwich-type electrochemiluminescence (ECL) aptasensor was designed.
• The in situ generated proline and PAMAM was served as coreactant for Ru.
• With triple signal amplification, the aptasensor could respond to 5.0 fM thrombin.

Here, an ultrasensitive electrochemiluminescence (ECL) aptasensor using in situ generated proline and polyamidoamine (PAMAM) dendrimers as coreactant for bis(2,2ʹ-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.