Peng Zhang

Exosomes are cell-derived nano-sized vesicles that have been recently recognized as new mediators for many cellular processes and potential biomarkers for non-invasive disease diagnosis and the monitoring of treatment response. To better elucidate the biology and clinical value of exosomes, there is a pressing need for new analytical technologies capable of the efficient isolation and sensitive analysis of such small and molecularly diverse vesicles. Herein, we developed a microfluidic exosome analysis platform based on a new graphene oxide/polydopamine (GO/PDA) nano-interface. To the best of our best knowledge, we report for the first time, the GO-induced formation of a 3D nanoporous PDA surface coating enabled by the microfluidic layer-by-layer deposition of GO and PDA. It was demonstrated that this nanostructured GO/PDA interface greatly improves the efficiency of exosome immuno-capture, while at the same time effectively suppressing non-specific exosome adsorption. Based on this nano-interface, an ultrasensitive exosome ELISA assay was developed to afford a very low detection limit of 50 μL(-1) with a 4 log dynamic range, which is substantially better than the existing methods. As a proof of concept for clinical applications, we adapted this platform to discriminate ovarian cancer patients from healthy controls by the quantitative detection of exosomes directly from 2 μL plasma without sample processing. Thus, this platform could provide a useful tool to facilitate basic and clinical investigations of exosomes for non-invasive disease diagnosis and to aid precision treatment.

Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe(3)O(4)@mTiO(2) microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO(2) layer, high specific surface area (167.1 m(2)/g), large pore volume (0.45 cm(3)/g), appropriate and tunable pore size (8.6-16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe(3)O(4)@mTiO(2) microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe(3)O(4)@mTiO(2) possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe(3)O(4)@TiO(2) microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe(3)O(4)@mTiO(2) composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

An integrated, 3D nano-engineered exosome profiling platform (ExoProfile chip) was developed to afford ultrasensitive, multiplexed detection of a panel of surface protein markers on circulating exosomes with a minuscular volume of plasma.