Isolation and Characterization of Exosomes from Cancer Cells Using Antibody-Functionalized Paddle Screw-Type Devices and Detection of Exosomal miRNA Using Piezoelectric Biosensor

Abstract

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 μM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

Keywords: MCF-7 cell line; exosome; immunoaffinity separation; microRNA; paddle screw; surface acoustic wave (SAW) biosensor.

Figure 1

(a) Top view of the dual-type SAW sensor and a picture of the packaged SAW sensor. (b) Measured S₂₁ of the SAW sensor fabricated on the LiTaO₃ substrate in air at 25 °C.

Figure 2

Various shapes of 3D paddle structures for the isolation of exosomes. The paddle screw shown in the red box was selected considering that it was convenient to manufacture with 3D printing and that it matched well with a 1.5 mL microtube.

Figure 3

(a) A custom-made paddle screw-rotating device. The tubes located at the bottom of the device consisted of one exosome containing cell-free supernatant, three PBS buffer solutions, and one DTT solution. (b) A graphical scheme of the principle of exosome isolation by paddle screw devices.

Scheme 1

Schematic representation of sandwich hybridization and gold staining signal amplification in the working sensor for the detection of miR-106b using the SAW biosensor (the reactions occurring in the reference sensor are shown in small boxes).

Figure 4

(a) Working sensor responses due to Hg²⁺-based hairpin loop formation, sandwich hybridization, and the subsequent gold staining reagent. A decrease in frequency indicates an increase in the effective mass of the sensor chip. A 1.0 nM concentration of the synthetic miR-106b was used in this experiment. (b) TEM images of AuNPs (left) and size-enhanced AuNPs by gold staining (right).

Figure 5

(a) Variations in the working sensor responses (solid line) and normalized sensor responses (dashed line) according to miR-106b concentrations. (b) Selectivity of the SAW biosensor toward miR-106b in comparison with the three other miRNA samples (10 μM).

Figure 6

Characterization of exosomes. (a) NTA and BCA total protein analysis of isolated exosomes from MCF-7 cells, (b) particle size distribution of isolated exosomes by PSs-COMB, and (c) Western blotting with antibodies against CD9 and CD81 for isolated exosomes by PSs-COMB.

Figure 7

(a) Extrapolation of the C_T values obtained from the RT-qPCR assay as a function of total RNA concentration. (b) Variations in the working sensor responses (solid line) and normalized sensor responses (dashed line) of SAW biosensors as a function of total RNA concentration.