▎ 摘　　要

NOVELTY - Preparing electrochemical luminescence sandwich biosensor comprises: (i) preparing gold nanoparticle-graphene modified glassy carbon electrode; (ii) preparing gold nanoparticle; (iii) labeling TBA2 with Ru(bpy) 2(dcbpy)NHS; (iv) pre-treating of Ru-TBA2 beacon with tris(2-carboxyethyl)phosphine and acetate buffer solution; (v) placing gold nanoparticle in glass bottle, treating, adding Ru-TBA2, incubating, adding tris-acetate buffer solution and sodium chloride, cultivating, and (vi) soaking step (i) resultant in in sulfhydryl-containing modification TBA1 solution and incubating. USE - The electrochemical luminescence sandwich biosensor is useful for detecting thrombin(claimed). ADVANTAGE - The sensor is simple to operate, fast in detection and not easy to be interfered by other substances and has good stability, sensitivity, repeatability and strong specificity of thrombin. DETAILED DESCRIPTION - Preparing electrochemical luminescence sandwich biosensor comprises: (i) preparing gold nanoparticle-graphene modified glassy carbon electrode by potentiostatic method; (ii) preparing gold nanoparticle (AuNPs) by citric acid reduction chloroauric acid method; (iii) labeling TBA2 with Ru(bpy) 2(dcbpy)NHS, dissolving in phosphate buffered saline solution to obtain a Ru-TBA2 beacon; (iv) pre-treating of Ru-TBA2 beacon with tris(2-carboxyethyl)phosphine and acetate buffer solution of pH 5.2 for 1 hour; (v) placing gold nanoparticle in a glass bottle, treating with sodium hydroxide, adding step (iv) Ru-TBA2, incubating at room temperature for 16 hours, adding pH 8.2 tris-acetate buffer solution and sodium chloride, cultivating for 24 hours under shading greenhouse, centrifuging to remove excess Ru-TBA2 and dissolving resulting Ru-TBA2-AuNPs in phosphate buffered saline buffer solution; and (vi) soaking gold nanoparticle-graphene modified glassy carbon electrode in a sulfhydryl-containing modification TBA1 solution for 2-8 hours, incubating with thrombin solution for 20 minutes and incubating with step (v) Ru-TBA2-AuNPs for 20 minutes. INDEPENDENT CLAIMS are also included for: (1) electrochemical luminescence sandwich biosensor prepared as mentioned above; and (2) use method electrochemical luminescence sandwich biosensor in detecting thrombin comprising (1) soaking biosensor in thrombin solution to be tested for 20-40 minutes, rinsing the electrode with phosphate buffer solution, (2) soaking of biosensor in 1-10 M Ru-TBA2-AuNPs for 20-40 minutes, rinsing electrode with a phosphate buffer solution, (3) using electrochemiluminescence biosensor as working electrode in the three electrode system, carrying out electrochemical luminescence detection using 0.05-0.1 M tri-n-propylamine solution as detection solution.