The proteins expressed on exosomes have emerged as promising liquid-biopsy biomarkers for cancer diagnosis. However, molecular profiling of exosomal proteins remains technically challenging. Herein, we report a nanozyme-assisted immunosorbent assay (NAISA) that enables sensitive and rapid multiplex profiling of exosomal proteins. This NAISA system is based on the installation of peroxidase-like nanozymes onto the phospholipid membranes of exosomes, thus avoiding the need for post-labelling detection antibodies. The exosomal proteins are determined by a sensitive nanozyme-catalyzed colorimetric assay less than 3 h, without the need for multi-step incubation and washing operations. Using NAISA to profile exosomal proteins from different cell lines and clinical samples, we reveal that tumor-associated exosomal proteins can serve as promising biomarkers for accurate cancer diagnosis in a cooperative detection pattern. Methods: Exosomes were engineered with DSPE-PEG-SH through hydrophobic interaction, and then were assembled with gold nanoparticles (2 nm) to produce Exo@Au nanozyme. The proteins on Exo@Au could be selectively captured by their specific antibodies seeded into a 96-well plate. The immobilized Exo@Au shows peroxidase-like activity to perform colorimetric assays by reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2 O 2 . The protein levels of exosomes were recorded on a microplate reader. Results: The NAISA platform is capable of profiling multiple exosomal proteins from both cancer cell lines and clinical samples. The expression levels of exosomal proteins, such as CD63, CEA, GPC-3, PD-L1 and HER2, were used to classify different cancer cell lines. Moreover, the protein profiles have been applied to differentiate healthy donors, hepatitis B patients, and hepatic cell carcinoma (HCC) patients with high accuracy. Conclusion: The NAISA nanozyme was allowed to rapidly profile multiple exosomal proteins and could have great promise for early HCC diagnosis and identification of other cancer types.

Hydrogen peroxide (H2O2) is widely involved in various physiological or pathological processes such as cell differentiation, proliferation, tumorigenesis, and immune responses. The accurate detection of H2O2 is highly required in many situations ranging from chemical sensing to biomedical diagnosis. However, it is exceedingly challenging to develop a single sensor that can respond to H2O2 in different conditions. Herein, a three-in-one stimulus-responsive nanoplatform (Au@MnO2@Raman reporter) was designed for colorimetry/SERS/MR tri-channel H2O2 detection which satisfied different applications. The MnO2 shell acted as a distance mediator between the gold nanoparticle (Au NP) core and the Raman reporter layer. In the presence of H2O2, the MnO2 shell is degraded, thus releasing the Mn2+ and Au NP core, which act as magnetic resonance (MR) and colorimetry signals, respectively. Simultaneously, the Raman reporters adsorb on the exposed Au NPs, resulting in the surface-enhanced Raman scattering (SERS) effect. The Au NP-based colorimetric assay was employed as H2O2 sensors for glucose detection while the turn-on signals of SERS and MR were used for H2O2 sensing and imaging in live cells and tumors, showing great versatility and flexibility of the multichannel probes in diverse situations.

Exosomes have recently emerged as some of the most promising biomarkers for disease diagnosis. Due to their small sizes and composition heterogeneity, exosomes are difficult to detect by currently available platforms. Here, we report a pHmediated assembly system that converts single nanosized exosomes into microsized clusters, which can be directly analyzed by conventional flow cytometry (FCM), breaking through the size limit of exosome analysis. We demonstrated the clinical utility of the pH-mediated clustering system by profiling the exosomal proteins from blood plasma samples of 33 cancer patients and 11 benign controls. The results indicated that the combination of MUC-1 and PD-L1 could serve as a new biomarker panel for the early diagnosis of liver cancer with high clinical accuracy. This pHmediated assembly strategy allows rapid, sensitive, and high-throughput analysis of exosome biomarkers by conventional FCM, which can be easily refined for use in both basic and clinical settings.

Exosomes, ranging from 30–150 nm in diameter, have emerged as promising non‐invasive biomarkers for the diagnosis and prognosis of numerous diseases. However, current research on exosomes is largely restricted by the lack of an efficient method to isolate exosomes from real samples. Herein, the first stimuli‐mediated enrichment and purification system to selectively and efficiently extract exosomes from clinical plasma for high‐throughput profiling of exosomal mRNAs as cancer biomarkers is presented. This novel isolation system relies on specific installation of the stimuli‐responsive copolymers onto exosomal phospholipid bilayers, by which the enrichment and purification are exclusively achieved for exosomes rather than the non‐vesicle counterparts co‐existing in real samples. The stimuli‐mediated isolation system outperforms conventional methods such as ultracentrifugation and polyethylene glycol‐based precipitation in terms of isolation yield, purity, and retained bioactivity. The high performance of the isolation system is demonstrated by enriching exosomes from 77 blood plasma samples and validated the clinical potentials in profiling exosomal mRNAs for cancer diagnosis and discrimination with high accuracy. This simple isolation system can boost the development of extracellular vesicle research, not limited to exosomes, in both basic and clinical settings.

Circulating tumor microenvironment-derived extracellular vesicles (cTME-EVs) are gaining considerable traction in cancer research and liquid biopsy. However, the study of cTME-EVs is largely limited by the dearth of a general isolation technique to selectively enrich cTME-EVs from biological fluids for downstream analysis. In this work, we broke through this dilemma by presenting a double-switch pH-low insertion peptide (D-S pHLIP) system to exclusively harvest cTME-EVs from the blood serum of tumor mouse models. This D-S pHLIP system consists of a highly sensitive pH-driven conformational switch (p K a ≈ 6.8) that allows specific installation of D-S pHLIP on the EV membranes in TME (pH 6.5 to 6.8) and a unique hook-like switch to “lock” the peptide securely on the cTME-EVs during the systemic circulation. The D-S pHLIP-anchored cTME-EVs were magnetically enriched and then analyzed with high-resolution messenger RNA sequencing, by which more than 18 times the number of TME-related differentially expressed genes and 10 times the number of hub genes were identified, compared with those achieved by the gold-standard ultracentrifugation. This work could revolutionize basic TME research as well as clinical liquid biopsy for cancer.