Amplified Thrombin Aptasensor Based on Alkaline Phosphatase and Hemin/G-Quadruplex-Catalyzed Oxidation of 1-Naphthol

An alkaline phosphatase (ALP)-based biosensor can in situ generate an electroactive product by enzymatic hydrolysis of inactive substrates. To obtain a higher signal-to-background ratio, a chemical redox cycling signal-amplified strategy based on the addition of a strong reducing agent has often be applied in the construction of ALP-based biosensors. However, the strong reducing agent not only affects the activity of ALP but also readily reacts with dissolved oxygen, leading to inaccurate results. In this work, a new signal-amplified strategy for a thrombin (TB) aptasensor based on the catalytic oxidation of ALP-generated products, 1-naphthol (NP), using hemin/G-quadruplex DNAzymes was reported. We implemented gold-nanoparticle-decorated zinc oxide nanoflowers (Au-ZnO) as the matrix for immobilizing ALP and TB aptamer (TBA) and then labeled it with hemin to form hemin/G-quadruplex/ALP/Au-ZnO bioconjugates (TBA II bioconjugates). Through a "sandwich" reaction, TBA II bioconjugates were captured on the electrode surface. The amplified signal was carried out in two steps: (i) an ALP-catalyzed inactive substrate, 1-naphthyl phosphate (NPP), in situ produces NP on the surface of the electrode; (ii) on the one hand, NP as a new reactant could be directly electrooxidized and generated an electrochemical signal, but, on the other hand, NP could be oxidized by hemin/G-quadruplex in the presence of H2O2, resulting in amplification of the electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM to 30 nM with a detection limit of 0.37 pM (defined as S/N = 3).