In Situ Generation of Self-Enhanced Luminophore by β-Lactamase Catalysis for Highly Sensitive Electrochemiluminescent Aptasensor

This work described a new electrogenerated chemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin (TB) based on the in situ generating self-enhanced luminophore by beta-lactamase catalysis for signal amplification. Briefly, a ruthenium complex (Ru-Amp), including two regions of [Ru(phen)(2)(cpaphen)](2+) and ampicillin (Amp), was synthesized as a self-enhanced ECL luminophore, which can produce an ECL signal through intramolecular interactions. Then, carbon nanotubes (CNTs) were used for immobilization of Ru-Amp via pi-pi stacking interactions to form the Ru-Amp@CNTs nanocomposite. Using poly(ethylenimine) (PEI) as a linkage reagent, Au nanocages (AuNCs), owing to their electronic property and large surface areas, were decorated to the CNTs to form the Ru-Amp@CNTs-PEI-AuNCs nanocomposites, which were further used to immobilize thrombin binding aptamer II (TBA II) to form a signal probe (Ru-Amp@CNTs-PEI-AuNCs-TBA II). Through sandwich tactics, TBA II bioconjugates, TB and TBA I were immobilized onto the gold nanoparticles modified electrode. Then, with the enzyme catalysis of beta-lactamase, a novel self-enhanced ECL luminophore (Ru-AmpA) was in situ produced, which could exhibit a significant enhancement of ECL signal, due to the structure transformation of an amide bond into a secondary amine. A sandwich ECL assay for TB detection was developed with excellent sensitivity of a concentration variation from 1.0 fM to 1.0 pM and a detection limit of 0.33 fM. Therefore, the self-enhanced ECL luminophore, combining the further enhancement by in situ enzymatic reaction, is expected to have potential applications in biotechnology and clinical diagnosis.