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

Liyan Kong; Jiaming Zhu; Wen Wang; Lehe Jin; Yanjiao Fu; Bohui Duan; Liang Tan

doi:10.1016/j.saa.2016.10.022

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

Spectrochim Acta A Mol Biomol Spectrosc. 2017 Feb 15:173:675-680. doi: 10.1016/j.saa.2016.10.022. Epub 2016 Oct 17.

Authors

Liyan Kong¹, Jiaming Zhu¹, Wen Wang¹, Lehe Jin², Yanjiao Fu², Bohui Duan², Liang Tan³

Affiliations

¹ Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China.
² Department of Chemistry, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China.
³ Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China; Department of Chemistry, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China. Electronic address: liangtan@hunnu.edu.cn.

PMID: 27780127
DOI: 10.1016/j.saa.2016.10.022

Abstract

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.

Keywords: Acridine orange; Fluorescence resonance energy transfer; Gold nanocomposites; Immunoreaction; Thrombomodulin.

MeSH terms

Acridine Orange / chemistry
Antibodies, Monoclonal / chemistry
Antibodies, Monoclonal / immunology
Fluorescence Resonance Energy Transfer / methods*
Fluorescent Dyes / chemistry
Gold / chemistry*
Human Umbilical Vein Endothelial Cells
Humans
Hydrogen Peroxide / pharmacology
Limit of Detection
Metal Nanoparticles / chemistry*
Microscopy, Electron, Transmission
Sensitivity and Specificity
Serum Albumin, Bovine
Spectrophotometry, Ultraviolet
Thrombomodulin / analysis*
Thrombomodulin / immunology

Substances

Antibodies, Monoclonal
Fluorescent Dyes
THBD protein, human
Thrombomodulin
Serum Albumin, Bovine
Gold
Hydrogen Peroxide
Acridine Orange