Nanoarray Enabled Size-Dependent Isolation and Proteomics Profiling of Small Extracellular Vesicle Subpopulations toward Accurate Cancer Diagnosis and Prognosis.

Small extracellular vesicles (sEVs) have emerged as noninvasive biomarkers in liquid biopsy due to their significant function in pathology and physiology. However, the phenotypic heterogeneity of sEVs presents a significant challenge to their study and has significant implications for their applications in liquid biopsies. In this study, anodic aluminum oxide films with different pore sizes (AAO nanoarray) were introduced to enable size-based isolation and downstream proteomics profiling of sEV subpopulations. The adjustable pore size and abundant Al3+ on the framework of AAOs allowed size-dependent isolation of sEV subpopulations through nanoconfined effects and Lewis acid-base interaction between AAOs and sEVs. Benefiting from the strong concerted effect, the simple AAO nanoarray enabled specific isolation of three sEV subpopulations, termed "50", "90", and "150 nm" groups, from 10 µL of complex biological samples within 10 min with high capture efficiencies and purities. Moreover, the nanopores of AAOs also acted as nanoreactors for comprehensive proteomic profiling of the captured sEV subpopulations to reveal their heterogeneity. The AAO nanoarray was first investigated on sEVs from a cell culture medium, where sEV subpopulations could be clearly distinguished, and three traditional sEV-specific proteins (CD81, CD9, and FLOT1) could be identified by proteomic analysis. A total of 3946, 3951, and 3940 proteins were identified from 50, 90, and 150 nm sEV subpopulations, respectively, which is almost twice the number compared to those obtained from the conventional approach. The concept was further applied to complex real-case sample analysis from prostate cancer patients. Machine learning and gene ontology (GO) information analysis of the identified proteins indicate that different-sized sEV subpopulations contain unique protein cargos and have distinct cellular components and molecular functions. Further receiver operating characteristic curve (ROC) analysis of the top five differential proteins from the three sEV subpopulations demonstrated the high accuracy of the proposed approach toward prostate cancer diagnosis (AUC > 0.99). More importantly, several proteins involved in focal adhesion and antigen processing and presentation pathways were found to be upregulated in prostate cancer patients, which may serve as potential biomarkers of prostate cancer. These results suggest that the sEV subpopulation-based AAO nanoarray is of great value in facilitating the early diagnosis and prognosis of cancer and opens a new avenue for sEVs in liquid biopsy.

Simultaneous quantification of exosomal MMP14 expression and proteolysis activity on a spherical dual-probe-based fluorescent nanosensor.

Among various exosomal proteins, matrix metalloproteinases (MMPs) are a family of membrane associated endopeptidases and have been considered as potential biomarkers in liquid biopsy owing to their multiple roles in pathological processes. However, the potential of MMP14 expression (MMP14-E) and MMP14 proteolytic activity (MMP14-A) in clinical diagnosis is still not clear due to the lack of sensitive and simultaneous detection techniques. Herein, we propose a fluorescent nanosensor for the simultaneous detection of MMP14-E and MMP14-A using a spherical aptamer/peptide dual-probe strategy. The aptamer and peptide probes were sequentially immobilized on Fe3O4 magnetic nanoparticles coated with gold nanoparticles (m-AuNPs) using a disulfide linker. MMP14 can be specifically recognized by the aptamer, while the proteolytic-active MMP14 can cleave the peptide probe. While achieving simultaneous detection, the proposed sensor provides better analytical performances than traditional MMP14 sensors owing to the m-AuNP-based spherical dual-probe strategy. This sensor has been successfully applied for the detection of exosomal MMP14 from cell culture media and real serum samples. Levels of both MMP14-E and MMP14-A increase in serum from cancer patients, indicating their potential applications as biomarkers in liquid biopsy for disease diagnosis and real-time surveillance.

Efficient exosome subpopulation isolation and proteomic profiling using a Sub-ExoProfile chip towards cancer diagnosis and treatment.

Exosomes have been extensively studied as liquid biopsy biomarkers in the past decade. However, the origin and molecular heterogeneity of exosomes hinder the research development moving from proof-of-concept to clinical applications. Herein, we report an integrated microfluidic platform termed Sub-ExoProfile chip, to achieve the selective isolation and subsequent proteomic profiling of multiplex exosome subpopulations simultaneously. The Sub-ExoProfile chip comprises three cylindrical self-assembled nanopillars, on which specific exosome capture antibodies (CD81, EpCAM, HER2) were immobilized to capture and sort different exosome subpopulations. It is worth noting that the 3D porous nanopillars afford enhanced interface binding efficiency; thus, a tumor-specific exosome subpopulation with lowly-expressed surface marker was still isolated with satisfactory capture efficiency. Moreover, the amphiphilic mesoporous silica nanoparticle self-assembled nanopillars also served as a nanoreactor for the enrichment and in situ digestion of exosomal proteins, providing improved performance for the mass-spectrometry based molecular characterization of exosome subpopulations. The Sub-ExoProfile chip was investigated on standard exosome samples from different breast cancer cell lines. The isolation and quantitative detection of exosome subpopulations were in line with the molecular subtype of breast cancer cell lines, and the molecular makeup was confirmed using classic microplate ELISA. Clinical samples from HER2-positive and triple-negative breast cancer patients were also examined using the Sub-ExoProfile chip. The quantitative results of three exosome subpopulations distinguished the three subtypes of breast cancer significantly. Most importantly, the molecular characterization of three exosome subpopulations revealed that the distinct exosome subpopulation participated in a different signaling pathway and performed distinct biological functions. It is envisioned that the analysis of the exosome subpopulation on the Sub-ExoProfile chip will facilitate the screening of tumor-specific exosomal biomarkers and open a new avenue for exosome-based liquid biopsy.

Nanomaterials assisted exosomes isolation and analysis towards liquid biopsy.

Exosomes has attracted tremendous research interests as they are emerging as a new paradigm of liquid biopsy. Although the concentration of exosomes in blood is relatively abundant, there still exists various vesicle-like nanoparticles, such as microvesicles, apoptotic bodies. It's an urgent need to isolate and enrich exosomes from the complex contaminants in biofluid samples. Moreover, the expressing level of exosomal biomarkers varies a lot, which make the sensitive molecular detection of exosomes in high demand. Unfortunately, the efficient isolation and sensitive molecular quantification of exosomes is still a major obstacle hindering the further development and clinical application of exosome-based liquid biopsy. Nanomaterials, with unique physiochemical properties, have been widely used in biosensing and analysis aspects, thus they are thought as powerful tools for effective purification and molecular analysis of exosomes. In this review, we summarized the most recent progresses in nanomaterials assisted exosome isolation and analysis towards liquid biopsy. On the one hand, nanomaterials can be used as capture substrates to afford large binding area and specific affinity to exosomes. Meanwhile, nanomaterials can also be served as promising signal transducers and amplifiers for molecular detection of exosomes. Furthermore, we also pointed out several potential and promising research directions in nanomaterials assisted exosome analysis. It's envisioned that this review will give the audience a complete outline of nanomaterials in exosome study, and further promote the intersection of nanotechnology and bio-analysis.

All-in-One Strategy for Downstream Molecular Profiling of Tumor-Derived Exosomes.

In light of the significance of exosomes in cancer diagnosis and treatment, it is important to understand the components and functions of exosomes. Herein, an all-in-one strategy has been proposed for comprehensive characterization of exosomal proteins based on nanoporous TiO2 clusters acting as both an extractor for exosome isolation and a nanoreactor for downstream molecular profiling. With the improved hydrophilicity and inherent properties of TiO2, exosomes can be captured by a versatile nanodevice through the specific binding and hydrophilicity interaction synergistically. The strong concerted effect between exosomes and nanodevices ensured high efficiency and specificity of exosome isolation with high recovery and low contaminations. Meanwhile, highly efficient downstream proteomic analysis of the purified exosomes was also enabled by the nanoporous TiO2 clusters. Benefiting from the porous structure of the nanodevice, the lysed exosomal proteins are highly concentrated in the nanopore to achieve high-efficiency in situ proteolytic digestion. Therefore, the unique features of the TiO2 clusters ensured that all the complex steps about isolation and analysis of exosomes were completed efficiently in one simple nanodevice. The concept was first proved with exosomes from cell culture medium, where a high number of identified total proteins and protein groups in exosomes were obtained. Taking advantage of these attractive merits, the first example of the integrated platform has been successfully applied to the analysis of exosomes in complex real-case samples. Not only 196 differential protein biomarker candidates were discovered, but also many more significant cellular components and functions related to gastric cancer were found. These results suggest that the nanoporous TiO2 cluster-based all-in-one strategy can serve as a simple, cost-effective, and integrated platform to facilitate comprehensive analysis of exosomes. Such an approach will provide a valuable tool for the study of exosome markers and their functions.

Sensitive and fast beverage/fruit antioxidant evaluation by TiO2 -Au/graphene nanocomposites coupled with MALDI-MS.

RATIONALE: Because the ratio of tripeptide glutathione (GSH) to glutathione disulphide (GSSG) is tightly associated with the oxidative stress and antioxidant level of an organism, it is often considered to be an indicator of the redox states of cells. Therefore, developing an integrated protocol for rapid, efficient, and low-cost detection of GSH to measure antioxidant ability is of significant importance for the diagnosis of oxidative stress-associated diseases. METHODS: TiO2 -Au/graphene nanocomposites were synthesized to integrate the characteristics of graphene as matrix for MALDI-MS, gold NPs as selective probes for GSH and the photocatalytic property of TiO2 . Under UV-visible (UV-Vis) light irradiation, OH can be produced by the TiO2 -Au/G composites, which can oxidize GSH to form GSSG. When various antioxidants are introduced into the aforementioned system, OH can be scavenged, thereby leaving part of GSH in its reductive format. Based on the ratio of GSH/GSSG, the antioxidative capability of various beverages and fruits can be determined. RESULTS: TiO2 -Au/G composites were employed to enrich GSH, where 0.01 mg/mL GSH can be efficiently extracted by the nanocomposites from aqueous solution and detected by MS with high signal-to-noise ratio. The proposed strategy was applied to evaluate three often-used antioxidants: Vitamin C, Vitamin E, and ß-carotene; and demonstrated that the antioxidative ability of VC is the strongest. To further evaluate the feasibility of the proposed strategy for antioxidative ability evaluation of complex sample, commercial juices and fresh fruit were also studied. CONCLUSIONS: A novel strategy for sensitive and fast characterization of the antioxidative ability of various beverages and fruits was developed based on TiO2 -Au/G nanocomposites coupled with MALDI-MS. Copyright © 2016 John Wiley & Sons, Ltd.

Polydopamine Grafted Porous Graphene as Biocompatible Nanoreactor for Efficient Identification of Membrane Proteins.

Functional nanomaterials, used as nanoreactors, have shown great advantages in a variety of applications in biomedical fields. Herein, we designed a novel nanoreactor system toward the application in membrane proteomics by using polydopamine-coated nanoporous graphene foams (NGFs-PD) prepared by a facile in situ oxidative polymerization. Taking advantage of the unique 3-D structure and surface functionalization, NGFs-PD can quickly adsorb a large amount of hydrophobic membrane proteins dissolved in sodium dodecyl sulfonate (SDS)/methanol and hydrophilic trypsin in aqueous solution, and then confine the proteolysis in the nanoscale domains to fasten the reaction rate. Therefore, the current nanoreactor system combines the multifunctions of highly efficient solubilization, immobilization, and proteolysis of membrane proteins. With the nanoreactor, digestion of standard membrane proteins can be finished in 10 min. 893 membrane proteins were identified from human glioma cells (U251). All these superiorities indicate that the biocompatible NGFs-PD nanoreactor system is of great promise to facilitate high-throughput membrane proteomic analysis.

Multifunctional nanoreactor for comprehensive characterization of membrane proteins based on surface functionalized mesoporous foams.

An integrated protocol is proposed here for efficient analysis of membrane proteins based on surface functionalized mesoporous graphene foams (MGF). The inherent hydrophobic nature of MGF and surface modification with hydrophilic chitosan (CS) make it highly suitable for the enrichment of hydrophobic membrane proteins from organic solvent, while remaining well-dispersed in aqueous solution for subsequent proteolysis. Therefore, such a multifunctional reactor ensures a facile solvent adjustment route. Furthermore, as a chitosan modified nanoporous reactor, it also provides a biocompatible nanoenvironment that can maintain the stability and activity of enzymes to realize efficient in situ digestion of the enriched membrane proteins. The concept was first proved with a standard hydrophobic membrane protein, bacteriorhodopsin, where a high number of identified peptides and amino acid sequence coverage were achieved even at extremely low protein concentration. The mesoporous reaction system was further applied to the analysis of complex real-case proteome samples, where 931 membrane proteins were identified in triplicate analyses by 2D LC-MS/MS. In contrast, with in-solution proteolysis, only 73 membrane proteins were identified from the same sample by the same 2D LC-MS/MS. The identified membrane proteins by the MGF-CS protocol include many biomarkers of the cell line. These results suggest that the multifunctional MGF-CS protocol is of great value to facilitate the comprehensive characterization of membrane proteins in the proteome research.