Ratiometric fluorescence and colorimetry dual-mode assay based on manganese dioxide nanosheets for visual detection of alkaline phosphatase activity

Alkaline phosphatase (ALP) activity plays a crucial role in foods and varies greatly in livestock and dairy products; it is quite difficult to compatibly monitor the different activities in various complicated foods. Herein, we proposed a ratiometric fluorescence and colorimetry dual-mode assay based on manganese dioxide (MnO₂) nanosheets for the visualization of ALP activity in livestock serum and pasteurized milk samples compatibly. MnO₂ nanosheets could oxidize dopamine into green fluorescent polydopamine (PDA) nanoparticles, so to quench the red fluorescent quantum dots (QDs), or oxidize colorless 3,3’,5,5’-tetramethylbenzidine (TMB) into yellow TMBox. In the coexistence of ALP and 2-phospho-L-ascorbic acid, MnO₂ nanosheets were reduced into Mn²⁺ ions by the catalysate of ascorbic acid, failing to generate PDA nanoparticles, and therefore, recovering the fluorescence of QDs for ratiometric fluorescence detection of high-activity ALP, or weakening the oxidization of TMB for colorimetric detection of ultralow-activity ALP. The ratiometric fluorescence-colorimetry combination extended the linear range over three orders of magnitude (0.04–80 mU/mL), and lowered the detection limit down to 0.015 mU/mL, along with profuse ALP-dependent (fluorescence) color changes for visual detection of ALP activity. Excellent recognition selectivity for ALP was attained over possibly coexistent reducing substances. Furthermore, the endogenous ALP were detected ranging from 17.32 to 269.54 mU/mL in seven typical livestock sera, consistent with that measured by commercial ALP assay kit; the detection results for ALP in four pasteurized milks matched well with that by test paper. The developed dual-mode assay held great potential for rapid on-site visual determination of reductant-related analytes in complicated matrices.