Controllable bisubstrate multi-colorimetric assay based on peroxidase-like nanozyme and complementary colorharmonic principle for semi-quantitative detection of H 2 O 2 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 H 2 O 2 concentration. The peroxidase-like activity of PEG@Fe 3 O 4 NPs was used to catalyze the oxidations of TMB and DA by H 2 O 2 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 (H 2 O 2 ) 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 H 2 O 2 . The detection limit and sensitivity for H 2 O 2 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.