Bio-Inspired NanoVilli Chips for Enhanced Capture of Tumor-Derived Extracellular Vesicles: Toward Non-Invasive Detection of Gene Alterations in Non-Small Cell Lung Cancer

Abstract

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.

Purification of HCC-specific extracellular vesicles on nanosubstrates for early HCC detection by digital scoring

“…The passive mixing behavior of the flow-through EVs in EV Click Chips was simulated (Supplementary Fig. 3 ) via the combined use of computational fluid dynamics (CFD) and dissipative particle dynamics (DPD) models 24 , offering a theoretical explanation on how the configuration of the EV Click Chip results in the enhanced physical contact 42 between TCO-grafted HCC EVs and Tz-grafted SiNWS.…”

“…To understand the crucial roles of the embedded silicon nanowires in SiNWS, the herringbone features in a PDMS chaotic mixer, and click chemistry-mediated EV capture, we carried out control experiments ( Supplementary Fig. 6) using (i) the devices without embedded silicon nanowires in SiNWS or herringbone features in the PDMS chaotic mixer, and (ii) the devices based on immunoaffinity EV capture 24 (NanoVilli Chips), in parallel with EV Click Chips and the ultracentrifugation approach 44 . EV Click Chips exhibited a recovery yield of 82.7 ± 1.34% and recovery purity of 90.2 ± 6.2%, which were significantly higher than those observed for the controls (Fig.…”

“…Since the majority of EVs in circulation are not of tumor origin, high background noise makes analysis of total EVs of limited diagnostic power 23 . To overcome this issue, our group 24 and others 23,25,26 have been exploring various immunoaffinity-based approaches to purify tumor-derived EVs. In parallel, others have been examining the potential of EVs and their mRNA cargos for HCC detection 27 .…”

“…Moreover, the microfluidic chaotic mixer made of polydimethylsiloxane (PDMS) facilitates repeated physical contact 36 between SiNWS and the flow-through HCC EVs, further enhancing the performance of EV capture. In contrast to previous antibody-mediated EV capture 24 , a pair of highly reactive click chemistry motifs 37 i.e., tetrazine (Tz) and trans-cyclooctene (TCO), are grafted onto EV capture substrates (i.e., SiNWS, via surface modification) and HCC EVs (via TCO-capture agent conjugation), respectively. Subsequently, the click chemistry reaction between Tz-grafted SiNWS and TCO-grafted HCC EVs is rapid, specific, irreversible, and bioorthogonal 37 , resulting in immobilization of the HCC EVs with improved capture efficiency and reduced background.…”

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods

“…Chips that make use of the latter typically employ a support structure, to which capturing molecules are attached. This can be antibodies against tumor-derived markers as EpCAM [123], or exosome-specific surface proteins like CD9, CD63, and CD81 [124]. Another alternative target is Annexin V which binds phosphatidylserine, a component of the EV lipid layer [125].…”