A ligand-free up-conversion nanoplatform based on enzyme cascade amplification strategy for highly sensitive detection of alkaline phosphatase

TMB was oxidized by Ag+ to TMBox quenching the fluorescence of the ligand-free UCNPs based upon the IFE principle. However, when in the presence of ALP and AAP, ALP could catalyze the hydrolysis of AAP to produce AA. The generated AA could inhibit TMBox production at both ends of the reversible process resulting in the fluorescent recovery of ligand-free UCNP and the detection ALP signal is amplified through this cascade process. Based on this principle, the cascade signal amplification strategy mediated by AA is constructed. [Display omitted] •A fluorescent and colorimetric detection of ALP based on ligand-free UCNPs was proposed.•The high sensitivity of the detection system is achieved by ECSAm.•The fluorescent quenching mechanism of UCNPs by TMBox based on IFE is flexible.•The assay based on the reversible oxidation of TMB can be extended to detect more analytes.•LOD of the sensor s for ALP is 0.032 U/L, which is lower than that of some reported literatures. An upconversion signal readout platform for the detection of alkaline phosphatase (ALP) was constructed by combining the inner filter effect (IFE) and the cascade signal amplification strategy. First, 3, 3′, 5, 5′ -tetramethylbenzidine (TMB) is oxidized by Ag+ to the oxidative product (TMBox), which quenches the up-conversion luminescence (UCL) of the ligand-free up-conversion nanoparticles (UCNPs) through the IFE principle. However, when in the presence of ascorbic acid 2-phosphate (AAP) and ALP, AAP is catalytically hydrolyzed by ALP to produce ascorbic acid (AA), which could not only decrease TMBox formation by reducing Ag+, but also reduce the TMBox generated to TMB, thereby resulting in fluorescence recovery of UCNP. Under optimal experimental conditions, ALP was linearly correlated in the range of 0.15∼8 U/L with a LOD of 0.032 U/L (S/N=3). In addition, the developed method was applied to the detection of ALP in actual serum samples, and the detection result was close to that of the traditional pNPP-based standard assay, which showed that the UCNP-TMB nanosystem has great potential to accurately detect ALP in the clinical bioassay.