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Osteosarcoma is one of the most frequent primary sarcoma of bone among adolescents. Early diagnosis of osteosarcoma is the key factor to achieve high survival rate of patients. Nevertheless, traditional histological biopsy is highly invasive and associated with the risk of arousing tumor spread. Herein, we develop a method integrating microfluidics and surface-enhanced Raman spectroscopy (SERS) to isolate plasma-derived exosomes and profile multiple exosomal biomarkers for the diagnosis of osteosarcoma. The method showed highly efficient isolation of exosomes directly from human plasma and can profile exosomes based on protein biomarkers, with the detection limit down to 2 exosomes per μL. The whole assay can be performed in 5 h and only consumed 50 μL of plasma for one analysis. With the method, we analyzed the level of three protein biomarkers, i.e., CD63, vimentin (VIM) and epithelial cell adhesion molecule (EpCAM), on plasma-derived exosomes from 20 osteosarcoma patients and 20 heathy controls. Significantly higher levels of CD63, VIM and EpCAM were observed on plasma exosomes from the osteosarcoma patients compared to the healthy controls. Based on the level of the exosomal biomarkers, a classification model was built for the rapid diagnosis of osteosarcoma, with the sensitivity, specificity and accuracy of 100%, 90% and 95%, respectively. The proposed method does not require complex operations nor expensive equipment, and has great promise in clinical diagnosis of cancer as a liquid biopsy technique., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Osteosarcoma is the most frequent primary bone cancer, particularly among children and adolescents. The early diagnosis of osteosarcoma is significant for timely clinical treatment to reduce the mortality of patients. Exosomes play a significant role in intercellular communication and serve as promising biomarkers in liquid biopsy for the diagnosis and monitoring of tumors. Herein, we report the utility of surface-enhanced Raman scattering (SERS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of osteosarcoma. We firstly profiled the intrinsic SERS signals and MALDI-TOF mass fingerprints of different subgroups of extracellular vesicles (EVs) and the corresponding cells, demonstrating that the SERS signals and MALDI-TOF mass spectra of exosomes from different types of cells were more discriminative compared to those of large and medium EVs and the cells themselves. Then, we characterized plasma-derived exosomes of 15 osteosarcoma patients and 15 healthy volunteers using SERS and MALDI-TOF MS, revealing distinctive biochemical differences in the spectra. We further utilized a data fusion approach to combine the two types of spectroscopic techniques, differentiating osteosarcoma patients from healthy controls with higher precision than either technique. The results reveal that the non-invasive liquid biopsy method using SERS and MALDI-TOF MS fingerprinting of exosomes has great potential for rapid diagnosis of osteosarcoma.

• A size-dependent microfluidic chip combines two PMMA layers with serpentine channels and a PCTE membrane in between. • Cleaning efficiency of contaminants is > 97 % and the recovery rate of exosomes is > 80 %. • Compared to UC, this method has merits of low-cost, easy operation, small volume sample consumption, and fast processing. • Compared to other filtration-based methods, the tangential flow filtration can largely avoid microfluidic blockage. Exosomes play an important role in microenvironmental regulation, cellular communication, tumor progression/metastasis and prognosis. A key challenge in the study of exosomes is the isolation and purification of exosomes from contaminants. In this study, we develop a size-dependent microfluidic chip to isolate and purify exosomes from human blood by tangential flow filtration. The microfluidic chip combines symmetrically two polymethyl methacrylate (PMMA) layers with serpentine channels and a nanoporous polycarbonate track etched (PCTE) membrane in between. Due to the uniform pore size (∼100 nm in diameter) of the nanoporous PCTE membrane, protein contaminants can be cleaned with high efficiency (> 97 %), and exosomes trapped in the microfluidic chip can be collected by reverse elution with high recovery (> 80 %). The collected exosomes are subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis. Based on the fingerprint peaks of exosomes, it is demonstrated that plasma proteins are largely cleaned and only exosome proteins dominate the mass spectra. Compared to ultracentrifugation (UC) that is considered as the "gold standard" for exosome isolation and purification, the size-dependent microfluidic chip-based protocol shows higher protein cleaning efficiency and exosome recovery rate. The whole process takes <3 h, shorter than UC. Considering that microfluidic based methods can be automatically controlled, the current method is promising in large-cohort clinical usage. [ABSTRACT FROM AUTHOR]