Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles

Statement of Significance

Extracellular vesicles (EVs), heterogeneous and tiny (40 nm ~ 5 mm in diameter) membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication. However, due to their small size, current protocols for isolation of Extracellular vesicles are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. We have developed a paper-based platform that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of Extracellular vesicles , and requires sample volumes as low as 10 mL. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of Extracellular vesicles in the clinic and the research setting to help advance our understanding of Extracellular vesicles functions in health and disease.

Microfluidics and Nanofluidics, May 2014, Volume 16, Issue 5, pp 849-856.

Chihchen Chen, Bo-Ren Lin, Hsi-Kai Wang, Shu-Ting Fan, Min-Yen Hsu, Chao-Min Cheng.

Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, 300, Taiwan.

Taichung Veterans General Hospital, Taichung, 407, Taiwan.

Abstract

We present a paper-based immunoaffinity platform for separating subsets of extracellular vesicles (EVs). This method is inexpensive, robust, easy-to-use (easy-to-prepare), and compatible with downstream analyses, such as scanning electron microscopy (SEM), enzyme-linked immunosorbent assays (ELISA), and transcriptome analysis. Extracellular vesicles are small membranous vesicles shed from both healthy and diseased cells and contain nucleic acid and protein cargo. EVs have been increasingly recognized as a means of cell–cell communication and hold a great potential for clinical applications. However, current protocols for isolation of EVs are often lengthy, cumbersome and require expensive equipment. Here, we have isolated EVs from small volumes of both human serum and aqueous humor samples using paper-based immunoaffinity devices. Captured EVs were analyzed morphologically using SEM and appeared statistically different in size (p value < 2.4 × 10⁻²²) and circularity (p value < 3.6 × 10⁻⁹) between subsets of Extracellular vesicles bearing different surface markers. Assessing the amount of Extracellular vesicles captured using paper-based ELISA using antibodies conjugated to horseradish peroxidase to produce a colorimetric readout was accomplished within 10 min. RNAs contained in EVs can be extracted to provide information for disease management. These paper-based immunoaffinity devices, we believe, open opportunities for a wide range of applications in both basic biology and clinical medicine.

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Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles

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