Supersandwich-type electrochemiluminescenct aptasensor based on Ru(phen)32+ functionalized hollow gold nanoparticles as signal-amplifying tags

• Electrochemiluminescent (ECL) aptasensor based on the signal amplification of supersandwich structure was designed.
• Hollow gold nanospheres were used as carriers to immobilize TBA 2.
• Supersandwich structure was obtained though in situ HCR.
• Ru(phen)32+ as ECL luminophore was intercalated into the grooves of dsDNA with high affinity.

An electrochemiluminescent (ECL) aptasensor was fabricated and used for the amplified detection of thrombin (TB) based on DNA supersandwich structure. Herein, hollow gold nanospheres (HGNPs) were firstly employed as effective tag-carriers for the immobilization of detection aptamer (TBA 2) to form the HGNPs labeled TBA 2 (HGNPs-TBA 2). Subsequently, streptavidin (SA) was used to block the non-specific binding sites of HGNPs-TBA 2 as well as to supply binding sites, which could further introduce numerous initiator DNA strands (bio-S1) via biotin–streptavidin specific interaction. Next, bio-S1 could in situ trigger hybridization chain reaction (HCR) to create a long nicked double helices analogous (dsDNA) in the present of ssDNA S2 and ssDNA S3 (S3 is partially complementary to the S2) to obtain the DNA supersandwich structure. Furthermore, Ru(phen)32+, a well-known ECL luminophore, could be intercalated into the grooves of dsDNA (Ru-dsDNA) to form the Ru-dsDNA-SA-HGNPs-TBA 2 bioconjugate. As a result, the target of TB was sandwiched between Ru-dsDNA-SA-HGNPs-TBA 2 and TBA 1. In this strategy, numerous Ru(phen)32+ could be immobilized on the electrode based on the supersandwich structure, resulting in an increased ECL signal output. A supersandwich ECL assay for TB detection was developed with excellent sensitivity of a large concentration variation from 5 fM to 50 pM and a detection limit of 1.6 fM.