Chemiluminescence detection of protein in capillary electrophoresis using aptamer-functionalized gold nanoparticles as biosensing platform

•Aptamer-functionalized gold nanoparticles (AuNPs) serves as biosensing platform.•AuNPs exhibits excellent catalytical behavior on luminol–H2O2 system.•Aptamer with high specificity as a new capture reagent for protein detection.•Highly selective and ultrasensitive detection of thrombin was achieved by CE-CL.•The strategy was successfully applied for the analysis of human plasma. Highly sensitive and selective detection of disease-related proteins play critical roles in clinical practice and diagnostic assays. Herein, we proposed a highly selective and ultrasensitive chemiluminescence (CL) method for protein detection in capillary electrophoresis (CE) using aptamer-functionalized gold nanoparticles (AuNPs) as biosensing platform. In this protocol, AuNPs were synthesized and conjugated with aptamer to form AuNPs-aptamer. Using thrombin and thrombin binding aptamer as an initial proof-of-concept recognization pair, AuNPs-aptamer was linked to thrombin to produce an AuNPs-aptamer–thrombin complex. The resulted complex and unbound AuNPs-aptamer were separated in CE and detected with luminol–H2O2 CL system. The developed strategy produced an ultrasensitive detection of thrombin down to 13.5fmol/L (S/N=3) with a linear range from 0.033 to 66.0pmol/L. The application of the present protocol was demonstrated by analyzing thrombin in human plasma samples with the recoveries of 87.6–116.8%. This novel strategy has many outstanding merits including high specificity of aptamer, excellent catalysis behavior of AuNPs, high sensitivity of CL detection, and high separation efficiency of CE.