Study On The Enzymatic Properties Of Alkaline Phosphatase Heterodimer And Its Preliminary Application Research In Liquid Biopsy

Alkaline phosphatase heterodimers are highly expressed in tumor cell lines such as colorectal cancer,breast cancer,cervical cancer and liver cancer.However,due to the lack of effective and specific probes to identify alkaline phosphatase heterodimers and the difficulty to obtained pure alkaline phosphatase heterodimers,the enzymatic properties of alkaline phosphatase heteodimers were still unknown.In addition,its expression in tumor cells and the distribution in exosomes are also unknown.It is of important significance for its application research in the field of circulating tumor cells and exosomes.In this paper,we studied alkaline phosphatase heterodimers using aptamer BG2 as a tool,which specifically binds alkaline phosphatase heterodimer.Alkaline phosphatase heterodimer was isolated by aptamer BG2 as an affinity ligand.The enzyme properties of placenta-intestinal alkaline phosphatase heterodimers were investigated,including the optimal buffer solution,thermal stability and the effects of EDTA,metal ions(Zn^2+,Cu^2+,Co²⁺,Mn²⁺,Ca²⁺,Cr²⁺,Hg²⁺,Mg²⁺)and amino acids（leucine and phenylalanine）on the enzyme activities.It was found that the heterodimers had worse thermal stability than placental alkaline phosphatase,and better thermal stability than intestinal alkaline phosphatase.EDTA,Zn²⁺and Hg²⁺strongly inhibited the activity of the heterodimers,while Mg²⁺,Cr²⁺and Mn²⁺activated the enzyme activity.In order to expand the application,we further confirmed the binding specificity of aptamer BG2 to the cell lines expressing heterodimer of alkaline phosphatase by flow cytometry assay using aptamer BG2 and antibodies as molecular probes.We also confirmed alkaline phosphatase heterodimer a biomarker of colorectal cancer by comparing the circulating tumor-related material captured by magnetic nanoparticles modified with aptamer BG2 from the blood of healthy people and patients with colorectal cancer.We established a method for specific isolation of exosomes with recovery greater than 90%,optimized the conditions of buffer solution,and verified the feasibility for exosome isolation.In order to improve the affinity of aptamers,we synthesized two small molecules with boric acid group,and modified them on oligonucleotide by using click chemistry.Therefore we established a modification method for oligonucleotide with boric acid groups.