Determination Of Alkaline Phosphatase By Ratio Fluorescence Based On Lanthanide Complex Polymers

Lanthanide coordination polymers（Ln CPs）,which are composed of lanthanide ions(Ln³⁺)and organic ligands through self-assembly,have attracted more and more attention due to their unique optical properties,easy preparation,high stability and encapsulation of guest materials.In particular,the long luminescence lifetime eliminates the interference of autofluorescence and scattered light in biological systems,so it is widely used in the determination of anions,metal ions,biomolecules and p H in human serum.In this paper,several Ln CPs composites were designed and synthesized,and a ratio fluorescence probe was constructed to detect alkaline phosphatase activity.Ln³⁺has a strong affinity with the ligand,and can coordinate with carboxyl,amino and phosphoric acid groups in the ligand,and sensitize Ln³⁺to luminescence through"antenna effect".Most lanthanide complexes are prepared using synthetic organic molecules as chelators,and relatively few studies have used alkaline phosphatase（ALP）substrate L-ascorbate-2-phosphate（AAP）as Ln³⁺ligand,especially in the determination of enzyme activity.In this experiment,AAP and Tb³⁺were self-assembled to form AAP-Tb as the frame material to realize the ratio fluorescence detection of ALP.（1）Due to the wide absorption spectrum,large molar extinction coefficient and rich surface chemical properties,manganese dioxide nanosheets（Mn O₂ NS）can be FRET（fluorescence resonance energy transfer）with CDs,thus effectively quenching CDs fluorescence.CDs@Mn O₂ NS composite nanomaterials were synthesized by one step hydrothermal method loaded with blue luminous CDs on Mn O₂ NS nanosheets.When AAP-TB coordination polymer was introduced into the system,the structure of AAP-TB was destroyed and the luminescence of Tb³⁺was weakened due to the dephosphorylation of AAP by ALP,while the generated ascorbic acid（AA）was able to reduce Mn O₂ to Mn²⁺,and CDs was released into the solution.The FRET process was weakened and its fluorescence was restored.The fluorescence probe based on this method can accurately detect the activity of ALP.The linear range was 0.1-1.0 U/m L,and the detection limit was 0.0047 U/m L.In particular,the probe showed visually identifiable fluorescence Color changes during the detection of ALP.The integrated sensing platform built with smart phone APP（Color Analyzer）was successfully used for the accurate determination of ALP activity in human serum.（2）Ln CPs has excellent self-assembly performance and can encapsulate various guest molecules.In this experiment,Ui O-66-NH₂ was encapsulated into the network structure of AAP-Tb as a guest molecule,and then assembled into core-shell nanneedles Ui O-66-NH₂@AAP-Tb.The composite nanoparticles exhibit Tb³⁺luminescence due to the ligand-metal charge transfer effect（LMCT）in Ui O-66-NH₂.The addition of ALP caused the hydrolysis of AAP-Tb,which resulted in the weakened coordination ability of AAP with Tb³⁺,and the release of the coated Ui O-66-NH₂.The hydrolyzed products AA and PO₄^3-could effectively weaken the effect of LMCT,thus synergistically enhancing the fluorescence of Ui O-66-NH₂.Based on this,the ratio fluorescence sensing platform of ALP was constructed.The linear range was 0.05-0.6U/m L,and the detection limit was 0.018 U/m L.The sensor platform can also be used to evaluate the performance of ALP inhibitors,and the detection of ALP activity in human serum has obtained a good recovery rate.This work provides a simple and sensitive assay for the detection of ALP.（3）Based on the packaging performance of Ln CPs,terephthalic acid（PTA）was wrapped into the AAP-Tb network structure during the synthesis process to synthesize the PTA@AAP-Tb composite nanomaterial,which showed the characteristic emission of Tb³⁺at 550 nm.After the addition of ALP,AA produced can capture the O₂ dissolved in the solution at high temperature and generate·OH,which then reacts with PTA to form blue fluorescent 2-hydroxyterephthalic acid（PTA-OH）,resulting in the enhancement of the fluorescence of PTA-OH at 450 nm.The linear range of the rate-type fluorescence sensing platform is 0.05-1.0 U/m L,and the detection limit is 0.012U/m L.The sensor has high sensitivity,high selectivity and excellent anti-interference ability,so it is expected to be used in the determination of ALP in complex biological samples.