A hybridization chain reaction based assay for fluorometric determination of exosomes using magnetic nanoparticles and both aptamers and antibody as recognition elements.

Abstract

Exosomes represent a new generation of biomarkers for the early diagnosis of hepatic carcinoma. A fluorometric assay is presented that is based on the hybridization chain reaction and magnetic nanoparticles for the highly sensitive determination of exosome (from HepG2 cells). Antibody as the recognition element was modified on the surface of magnetic nanoparticles. Antibody was used to capture the exosome. The Probe 1 was consisted of aptamer sequence and trigger sequence. Aptamer sequence will bind with the surface protein of exosome. The trigger sequence will hybridize with the probe2 (FAM-labeled) and the probe3 (FAM-labeled). So the product of the hybridization chain reaction will present a strong fluorescence signal. The fluorescence product of hybridization chain reaction will link with magnetic nanoparticles through the 'magnetic nanoparticles-antibody-exosome-aptamer' structure. The product can be separated from the matrix due to the present of the magnetic nanoparticles. The excitation was set at 490nm. The fluorescence value of the emission spectra at 519nm was set as the signal response. The linear range of this assay is from 1000 to 107 particles·mL-1. The detection limit is 100 particles·mL-1. This assay was applied to the determination of exosome from the hepatic carcinoma cells. Graphical abstractIn the presence of exosmes, the hybridization chain reaction was triggered and strong greenfluorescence will be produced. Moreover, the magnetic particles can separate the products from the matrix.