Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays

• A turn-off, AuNP-catalyzed amplified colorimetric thrombin aptasensor is presented.
• Reductive bleaching of colored substrate is used as a novel indicator reaction.
• We explore its fundamental principle and amplification factor theoretically.
• Endpoint-based colorimetric assay was achieved using 4-NP as the substrate.
• Kinetic-based colorimetric assay was first achieved using MB as the substrate.

Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91 pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10 pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications.