Highly sensitive thrombin detection by matrix assisted laser desorption ionization-time of flight mass spectrometry with aptamer functionalized core–shell Fe3O4@C@Au magnetic microspheres

Here, we describe a sensitive and specific method for thrombin detection with aptamer functionalized core–shell Fe3O4@C@Au magnetic microspheres (Au-MMPs). Firstly, Au-MMPs were synthesized through surface adsorption of gold nanoparticles onto PDDA functionalized Fe3O4@C magnetic microspheres. Then, the as-synthesized Au-MMPs were developed as new substrate for immobilization of thrombin binding aptamer (TBA) through easy formation of Au–S bond. After that, the prepared aptamer functionalized Au-MMPs (TBA@Au-MMPs) were used as effective magnetic absorbent to extract trace level of thrombin from dilute solutions. Finally, enriched thrombin was digested by trypsin and analyzed by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the efficient affinity extraction ability of our TBA@Au-MMPs and the sensitive mass readout of MALDI-TOF, highly sensitive detection of thrombin was achieved. The limit of detection was as low as 18 fmol, corresponding to 0.36 nM thrombin in 50 μL original solution. Linear relation was observed within a concentration range from 0.5 nM to 10 nM with linear correlation R2 = 0.998. Other proteins including human serum albumin (HSA), Ig G, transferrin, oval albumin (OVA) and fetal calf serum did not interfere with thrombin detection. This simple method holds great potential for analyzing, sensing, purification and preconcentration of proteins in biological fluids.