Highly sensitive turn-on fluorescence detection of thrombomodulin based on fluorescence resonance energy transfer

As an integral glycoprotein on the surface of endothelial cells, thrombomodulin (TM) has very high affinity for thrombin. TM has been regarded to be a marker of endothelial damage since it can be released during endothelial cell injury. In this work, a highly sensitive fluorescence method for the quantitative detection of TM was developed. TM antibody (Ab) and bovine serum albumin (BSA) were bound on gold nanoparticles (AuNPs) to construct BSA-AuNPs-Ab nanocomposites and they were characterized by transmission electron microscope and UV-vis spectrophotometry. The fluorescence of acridine orange (AO) was quenched by the prepared gold nanocomposites based on fluorescence resonance energy transfer (FRET). In the presence of TM, the fluorescence was turned on due to the effective separation of AO from the surface of gold nanocomposites. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the logarithm of the TM concentration from 0.1pgmL^-1 to 5ngmL^-1 with a low detection limit of 12fgmL^-1. The release of soluble thrombomodulin (sTM) by the injured HUVEC-C cells in the presence of H₂O₂ was investigated using the proposed method. The released sTM content in the growth medium was found to be increased with the enhancement of contact time of the cells with H₂O₂.