Paper-Based Devices for Capturing Exosomes and Exosomal Nucleic Acids From Biological Samples

Abstract

Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.

Keywords: colorimetric sensing; exosomal miRNA; exosome; immunoassay; nucleic acid extraction; paper-based device; plasma; wound fluid.

FIGURE 1

The scheme describes the experimental design in this study. HCT116 cells were cultured in various microenvironments (pH 7.4 and 6.5), characterized by microscope, ultrafiltrated to collect concentrated exosomes, which were then analyzed by a qNano instrument to determine their concentration and size distribution, and subjected to paper-based immunoaffinity devices for exosome capture. The morphology of captured exosomes was subsequently characterized by SEM and their amount was quantified by ELISA. Eventually, exosomal nucleic acids were absorbed by paper-based silica devices for miR-21 quantification by RT-qPCR.

FIGURE 2

Characterization of HCT116 cells cultured in various microenvironments of (A)∼(D) pH 7.4 after (A) 1, (B) 2, (C) 3, and (D) 4 days, and (E)∼(H) pH 6.5 after (E) 1, (F) 2, (G) 3, and (H) 4 days by microscope. The scale bar is 100 µm.

FIGURE 3

Particle size distribution and concentration of (A) exosomes from HCT116 cell culture medium and (B) commercial lyophilized exosomes analyzed by qNano.

FIGURE 4

SEM pictures characterize surface morphology of (A) untreated sample, (B) commercial lyophilized exosomes, and (C,D) HCT116-derived exosomes captured by paper-based immunoaffinity devices. Red arrows indicate captured exosomes.

FIGURE 5

Quantification of exosomes performed by color intensity assessment including commercial lyophilized samples (blue columns) and exosomes captured from the medium of HCT116 cells cultured in various microenvironments of pH 7.4 (red column) and 6.5 (green column). Inset is the calibration curve of exosomes from commercial samples (blue columns). An asterisk (*) denotes a p value <0.05 (obtained from t-test). All of the results were obtained by P-ELISA.

FIGURE 6

Quantification of miR-21. (A) Calibration curve for standard miR-21 samples by paper-based silica devices and quantified by RT-qPCR. (B) miR-21 contents (estimated by Ct values) in exosomes from standard samples and from 10 to 20 ml pH HCT116 samples cultured in pH 7.4 and pH 6.5 microenvironments. Three asterisks (***) denote a p value <0.001 (obtained from t-test). (C) miR-21 concentrations (copies/µL) of 10 and 20 ml HCT116 samples cultured in pH 7.4 and pH 6.5 microenvironments, calculated from calibration line in (A) and corresponding Ct values in (B). (D) U6 SiRNA contents (estimated by Ct values) derived from HCT116 cells cultured in pH 7.4 (red column) and pH 6.5 (green column) conditions.

FIGURE 7

Quantification of exosomal miR-21 from bio-samples captured by paper-based system. (A) Plasma-derived samples were obtained by flowing platelet-poor plasma (PPP, pink column) through 200 nm- and 30 nm-membrane to collect Retention (blue column) and Filtrate (green column) samples, respectively. No template control (NTC) was for comparison and analysis. (B–D) Fluidic samples were obtained from lesions of patients before and after being monitored for (B) 3 days, (C), 7 days, and (D) 1 month.