Selective detection of exosomes by elemental labeling ICP-MS based on cholesterol-EpCAM aptamer proximity ligation mediated MNAzyme assembly

Exosomes, carrying cancer-related proteins derived from parental cells, provide a liquid biopsy probability for early cancer diagnosis. However, the direct analysis of tumor derived exosomes in complex human fluids is still challenging due to the matrix interferences and the low proportion of cancer derived exosomes. Herein, a cholesterol-epithelial cell adhesion molecule (EpCAM) aptamer-mediated proximity ligation assay triggering multicomponent nucleic acid enzyme (MNAzyme) assembly was proposed for detection of exosomes by lanthanide elemental labeling inductively coupled plasma mass spectrometry (ICP-MS). Specifically, the exosomes were recognized through EpCAM aptamer and cholesterol, allowing aptamerEpCAM-partzyme A and cholesterol-partzyme B to hybridize with each other, and assembling into the exosome-MNAzyme complex. With the presence of the cofactor Mn2+, the exosome-MNAzyme complex could catalyze the cleavage of the ribonucleobase-containing substrate deoxyribonucleic acid (DNA) on the Tb-substrate DNA-magnetic beads, releasing Tb tags as readout signal for ICP-MS detection. Due to the recognition of dual exosome surface biomarkers through the proximity probes, the selectivity for the detection of tumor derived exosomes was greatly improved. The developed approach was used for the direct analysis of exosomes in human plasma samples from oral squamous cell carcinoma (OSCC) patients and healthy volunteers, and the concentration of exosomes in the OSCC patient group was found to be significantly higher than that in the healthy group. It manifested that the proposed strategy can easily distinguish healthy volunteers from cancer patients by purification-free quantification of exosomes in human plasma, exhibiting good potential of the developed strategy in early cancer diagnosis.