Enrichment of exosomes with high structural and functional integrity is a critically important step before downstream applications in biological and clinical domains. In this work, dynamic biological interfaces functionalized fructose-responsive immunomagnetic beads (Fr-IMBs) were fabricated by incorporating the dynamic biological interfaces composed of dopamine and 4-carboxyphenylboronic acid with immunomagnetic beads. These fabricated Fr-IMBs were successfully applied to capture the MCF-7 exosomes with a high capture efficacy of 91.0%. After incubation with fructose, the molecular structure of the dopamine-boronic acid complexation can be rearranged and the antibody (anti-CD63) bound exosomes were released competitively, with a yield greater than 79.8%. Those exosomes that underwent both immunomagnetic separation and fructose triggered release remained their good bioactivity to promote MCF-7 cell migration. Better yet, exosomes enriched by Fr-IMBs possessed higher purity and better preservation of tumor-related molecular information of the exosomal proteins than those obtained through the ultracentrifugation approach, as proved by the comparative proteomics. These proposed sugar-responsive immunomagnetic beads may serve as biofriendly candidates in enriching useful biomarkers (e.g., cells, nucleic acids, proteins, etc.) efficiently from complex biological samples with high purity.
In recent years, exosomes as the intercellular vehicles have received extensive attention for their fundamental roles in biosystem and as novel biomarkers for early detection and therapeutic applications. It has been demonstrated that anti-CD63 can effectively target exosomes from cancer cells. In this work, multifunctional nanoelectrode-nanopore nanopipettes (MNNPs) were prepared to detect exosomes secreted from human cervical cancer cells (HeLa cells) at the single-entity level using the nanopore- based resistive pulse sensing technique. Synthetic liposomes were also measured for comparison. By modifying the anti-CD63 on the carbon nanoelectrode surface near the nanopore, the detected current signals were very different from the signals from the non-modified MNNPs. The differences were induced by the specific interactions between anti-CD63 and the membrane protein CD63 at the surface of exosomes. No obvious differences were observed when liposomes were tested. Furthermore, by utilizing micromanipulation technology, the position of the nanopipette tip could be accurately located, which enabled monitoring the secretion of exosomes from individual cells in a real-time manner. In the control experiment, the probe-to-cell distance was optimized and the HeLa cells incubated with exosomes inhibitor (GW4869) were tested by MNNPs. These significantly reduced translocation signals proved that anti-CD63 modified MNNPs were competent for exosomes detection in complex cellular environments.
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