A Magnetic-induced Electrochemical Aptasensor Based On Pb²⁺-dependent DNAzyme For The Detection Of Thrombin

Thrombin,a serine protease,is converted from prothrombin by the action of factor Xa and converts fibrinogen to fibrin.It plays an important role in the mechanism of coagulation cascade.Studies have shown that thrombin can not only accelerate the occurrence of venous thromboembolism and the progression of kidney disease but also participate in a variety of diseases such as central nervous system diseases.Therefore,it is important to quantitatively detect thrombin content in complex biological samples for clinical research and early diagnosis.In this study,combined with aptamer technology,nanotechnology and Pb²⁺-dependent DNAzyme-assisted signal amplification technology,a label-free magnetic-induced electrochemical aptamer sensor was proposed for the detection of thrombin.Based on magnetic biocomposites and Pb²⁺-dependent DNAzyme,an electrochemical sensing platform was constructed for the detection of thrombin.There were three nucleotide including strands complementary strand?S1?,thrombin aptamer?S2?and Pb²⁺-dependent DNAzyme?S3?.Magnetic biocomposites were obtained by modifying dsDNA on the surface of Fe₃O₄@Au nanoparticles?Fe₃O₄@Au-S1/S2 NPs?and finally adsorbed on the surface of magnetic glassy carbon electrode with magnetic field directed self-assembly.In the presence of thrombin,S2 competitively bonded with thrombin.Exposed S1 were cleaved with the help of S3,so that the methylene blue were adsorbed on the DNA by electrostatic action and a weak electrochemical signal is detected by differential pulse voltammetry.When thrombin was not present,the S1/S2 dsDNA was not destroyed.Therefore the downstream reaction could not proceed,and the dsDNA adsorbed a large amount of methylene blue,producing a distinct electrochemical signal.Thus,the thrombin detection could be recorded by monitoring the electrochemical signal reduction of methylene blue.It was convenient for magnetic separation and magnetic induction by introducing Fe₃O₄@Au NPs.Moreover,with the unique properties of aptamer and assistance of Pb²⁺-dependent DNAzyme,this method exhibited high selectivity and excellent sensitivity to thrombin.This method exhibited a high sensitivity toward thrombin with a broad linear range from 5 pmol L^-1 to 5 nmol L^-1 and a limit of detection of 1.8 pmol L^-1.The proposed method was successfully applied to the detection of thrombin in biological samples and satisfying recoveries were acquired.