Tyndall-effect-based colorimetric enzyme-linked aptamer assay for instrument-free thrombin detection via enzyme-triggered degradation of MnO2 nanosheets

In this work, an innovative Tyndall-effect-based (TE-based) colorimetric strategy was developed to detect the activity of alkaline phosphatase (ALP) via enzyme-triggered degradation of MnO₂ nanosheets (MnO₂ NSs), and it was further extended to construct a TE-based colorimetric enzyme-linked aptamer assay (ELAA) for monitoring thrombin (TB) concentrations. In the presence of ALP, ascorbic acid 2-phosphate (AAP) was hydrolyzed to ascorbic acid (AA), which in turn reduced MnO₂ NSs to Mn 2+ and led to their structural decomposition. Under the irradiation of a handheld laser pointer, the TE signal of the detection solution gradually diminished with increasing ALP concentration. The quantitative measurement of ALP was further achieved using a smartphone to read out the TE signal, with a limit of detection (LOD) of 0.35 mU/mL. Furthermore, this strategy effectively functioned as an exquisite signal generation mechanism to devise a TE-based colorimetric ELAA for TB detection. The results demonstrated that the proposed method exhibited high specificity and sensitivity for TB, capable of linear detection over a concentration range of 1–25 nM with a LOD of 0.38 nM (3σ). In actual human serum samples, the recovery of TB ranged from 93.40 to 109.30%. To our knowledge, this work presents the first TE-based colorimetric ELAA for instrument-free protein analysis by leveraging enzyme-triggered MnO₂ nanosheet degradation. This approach establishes a novel signal transduction mechanism for colorimetric ELAA, thereby offering significant potential to expand the theoretical scope of this assay format and broaden its applications in various resource-limited settings. Graphical abstract