Controllable bisubstrate multi-colorimetric assay based on peroxidase-like nanozyme and complementary colorharmonic principle for semi-quantitative detection of H2O2 with the naked eye.

Naked-eye semi-quantitative (NEQ) assays should exhibit vivid color variations and one-to-one correspondence between the analyte concentrations and the color display. Herein, we report a bisubstrate multi-colorimetric system, constituted by 3,3',5,5'-tetramethylbenzidine (TMB) and dopamine (DA), which carries out a controllable NEQ assay based on the complementary colorharmonic principle. This bisubstrate system is a universal threshold NEQ assay with tunable sensitivity and detection window depending on the H2O2 concentration. The peroxidase-like activity of PEG@Fe3O4 NPs was used to catalyze the oxidations of TMB and DA by H2O2 to the colored products. On the basis of UV-vis spectra data, it was speculated that the oxidation product of TMB (TMB·+) could oxidize DA in this system. The concentration of DA controls the consumption of oxidant (H2O2) and the oxidation of TMB. By controlling the molar ratio of TMB to DA, the bisubstrate system precisely showed multicolor displays (e.g., three-color display: orange, gray, and blue) at submillimolar and millimolar concentrations of H2O2. The detection limit and sensitivity for H2O2 were 0.4 mM and 0.1 mM, respectively. Next, the system was applied to the threshold detection of hypoglycemia (orange), normal (gray), and hyperglycemia (blue) in spiked samples on both gel- and paper-based test strips. Digitalized colorimetric results using the red-green-blue (RGB) analysis with smartphone application were achieved. This work provides a new strategy of multi-colorimetric assay that takes advantages of controllability, threshold detection, vivid color variations, and reproducibility (CVs were 1.1-2.1%), which could be potentially useful for in-field and point-of-care applications.