Molecular profiling of extracellular vesicles via charge-based capture using oxide nanowire microfluidics.

Extracellular vesicles (EVs) have shown promising features as biomarkers for early cancer diagnoses. The outer layer of cancer cell-derived EVs consists of organotropic metastasis-induced membrane proteins and specifically enriched proteoglycans, and these molecular compositions determine EV surface charge. Although many efforts have been devoted to investigating the correlation between EV subsets obtained through density-, size-, and immunoaffinity-based captures and expressed membrane proteins, understanding the correlation between EV subsets obtained through surface charge-based capture and expressed membrane proteins is lacking. Here, we propose a methodology to profile membrane proteins of EV subsets obtained through surface charge-based capture. Nanowire-induced charge-based capture of EVs and in-situ profiling of EV membrane proteins are the two key methodology points. The oxide nanowires allowed EVs to be obtained through surface charge-based capture due to the diverse isoelectric points of the oxides and the large surface-to-volume ratios of the nanowire structures. And, with the ZnO nanowire device, whose use does not require any purification and concentration processes, we demonstrated the correlation between negatively-charged EV subsets and expressed membrane proteins derived from each cell. Furthermore, we determined that a colon cancer related membrane protein was overexpressed on negatively charged surface EVs derived from colon cancer cells.

Microfluidic-based capture and release of cancer-derived exosomes via peptide-nanowire hybrid interface.

Cancer-derived circulating exosomes or nanoscale extracellular vesicles are emerging biomarkers for disease detection and treatment because of their cell-specific constituents and unique intercellular pathways. For efficient exosome isolation from bio-fluids, the design of high-affinity nanointerfaces is of great importance in the development of miniaturized systems for the collection of exosomes. Herein, we report peptide-functionalized nanowires as a biorecognition interface for the capture and release of cancer-derived exosomes within a microfluidic channel. Based on the amino-acid sequence of EWI-2 protein, a partial peptide that bound to the CD9 exosome marker and thus targeted cancer exosomes was screened. Linkage of the exosome-targeting peptide with a ZnO-binding sequence allowed one-step and reagent-free peptide modification of the ZnO nanowire array. As a result of peptide functionalization, the exosome-capturing ability of ZnO nanowires was significantly improved. Furthermore, the captured exosomes could be subsequently released from the nanowires under a neutral salt condition for downstream applications. This engineered surface that enhances the nanowires' efficiency in selective and controllable collection of cancer-derived exosomes provides an alternative foundation for developing microfluidic platforms for exosome-based diagnostics and therapeutics.

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires.

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.