Highly enhanced electrochemiluminescence based on pseudo triple-enzyme cascade catalysis and in situ generation of co-reactant for thrombin detection.

In this work, a novel pseudo triple-enzyme cascade catalysis amplification strategy was employed to fabricate a highly sensitive electrochemiluminescence (ECL) aptasensor for thrombin (TB) detection. The signal amplification of the proposed aptasensor was based on the synergistic catalysis of glucose dehydrogenase (GDH) and hemin/G-quadruplex to generate a co-reactant in situ for the ECL of peroxydisulfate. Gold nanorods (AuNRs) conjugated with GDH and hemin/G-quadruplex were used as the secondary aptamer bioconjugate (TBA II) in this aptasensor. TB was sandwiched between TBA II and a thiol-terminated TB aptamer which self-assembled on the AuNRs-modified electrode. The pseudo triple-enzyme cascade catalysis was completed as follows: firstly, GDH could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of β-nicotinamide adenine dinucleotide hydrate (NAD(+)) into β-nicotinamide adenine dinucleotide hydrogen (NADH). Then, the hemin/G-quadruplex acted as NADH oxidase, could rapidly oxidize NADH into NAD(+) accompanied with the generation of H2O2. Simultaneously, the hemin/G-quadruplex served as the horseradish peroxidase (HRP)-mimicking DNAzyme that further catalyzed the reduction of H2O2 to generate O2in situ. Then the O2 produced acted as the co-reactant of peroxydisulfate, resulting in significant ECL signal amplification and highly sensitive ECL detection. The proposed aptasensor showed a wide linear range of 0.0001-50 nM with a low detection limit of 33 fM (S/N = 3) for TB determination. The present work demonstrated that the novel strategy has great advantages of sensitivity, selectivity and reproducibility, which hold new promise for highly sensitive bioassays applied in clinical detection.