An acoustofluidic device for sample preparation and detection of small extracellular vesicles
image: (A) The acoustofluidic chip comprises a channel containing sharp-edge microstructures that can be acoustically activated with an acoustic buzzer. (B) In the absence of acoustic activation, no fluorescence signal is detected as sEVs pass through the channel in the detection region. (C) When the chip is acoustically activated, sEVs containing specific surface markers are concentrated at the tips of the sharp-edge microstructures and detectable by fluorescence microscopy. (D) Experimental demonstration of on-chip detection of sEVs based on their surface proteins. A fluorescent signal is observed when the target protein is present above the detection threshold, while no signal appears if the protein is absent. CD63 is used as a control biomarker due to its common presence on sEVs. Scale bar, 100 μm.
Credit: Jessica F. Liu, Duke University School of Medicine.
Small extracellular vesicles (sEVs, ~30–140 nm) circulate in many biofluids and carry genomic, proteomic, and metabolomic cargo, making them become a a powerful vector for liquid biopsy, enabling the noninvasive assessment of physiological and pathological states. Clinically, sEV analysis is often limited by tiny patient sample volumes, so methods must work efficiently at small scale. Yet common techniques struggle here: nano–flow cytometry needs bulky, expensive instruments and extra preprocessing, and Western blotting is slow and typically prevents recovery of intact sEV subpopulations for downstream work. Microfluidic platforms help by handling tiny volumes and integrating isolation, purification, and detection on a compact chip, but there’s still a need for simpler, faster, and more sensitive approaches that suit point-of-care use. “To address this, we develop an acoustofluidic strategy—combining acoustics with microfluidics—using sharp-edge microstructures driven by a small acoustic buzzer to concentrate antibody-bead–bound sEVs for immediate on-chip fluorescence readout, even from ~50 µL samples.” said the author Jessica F. Liu, a researcher at Duke University School of Medicine, “The intended gap this fills is a portable, low-cost, easy-to-use platform for rapid, specific detection of sEV subpopulations at the point of care, with potential to expand into multiplexed, organ-specific monitoring.”
The acoustofluidic device comprises a polydimethylsiloxane (PDMS) layer with a microchannel containing sharp-edge microstructures on a glass substrate to which an acoustic wave generator is adhered. The fluid channel with sharp-edge structures was fabricated using soft lithography techniques [41]. Briefly, PDMS (Ellsworth) was molded using an SU-8/silicon wafer pattern to form a 100-μm-tall × 800-μm-wide microchannel containing 400-μm-long sharp-edge microstructures spaced at 400-μm intervals on alternating sides of the channel. The channel was then plasma bonded onto a 25 mm × 50 mm glass slide (Fisher) to form the base of the channel. Finally, an acoustic buzzer (Digikey, No. 668-1407-ND) capable of activating the sharp-edge microstructures was adhered to the glass slide at one end of the microchannel using clear epoxy (Amazon).
The following experimental results demonstrate the effectiveness of the device. In the size-selectivity validation experiment, when the acoustic field is on, 5-µm beads rapidly accumulate at the tips of the device’s sharp-edge structures, while 400-nm nanoparticles/unbound sEVs remain dispersed; when the acoustic field is off, neither population aggregates, and this phenomenon can be repeatedly observed at multiple sharp-edge sites. For the streptavidin–biotin binding quantification and detection efficiency, under a 4 kHz, 1.31 W square-wave drive, complexes of 5-µm streptavidin beads and 200-nm biotin nanoparticles are captured at the tips and exhibit pronounced fluorescence; the FIR (tip-to-background fluorescence intensity ratio) increases monotonically with bead concentration (10¹–10⁴ µL⁻¹); in the absence of streptavidin beads or without the acoustic field, FIR ≈ 1. Finally, in the marker capture and detection experiment, when anti-EGFR beads were co-incubated with HeLa cells (EGPR-positive [sic]), the FIR was 6.00 ± 0.46, whereas with anti-EGFR beads co-incubated with K562 cells (EGPR-positive [sic]), the FIR was 1.01 ± 0.03, indicating that the device can specifically capture sEVs.
The acoustofluidic technology described in this article enables highly flexible, specific, and efficient capture and detection of circulating extracellular vesicles (sEVs) from small sample volumes. Its portability, low cost, and ease of use make it an ideal tool for point-of-care detection of sEV surface markers, while its modular design allows for one-step, high-throughput capture and detection of diverse sEV populations. Additionally, the system’s ability to be easily parallelized enables simultaneous, multi-marker analysis with high precision. Future applications include the capture and detection of organ-specific sEVs from peripheral blood, enabling minimally invasive, multi-omic disease assessment beyond cancer biomarkers. “Ongoing work will focus on multiplexing the device for the simultaneous detection of multiple targets, further expanding its diagnostic capabilities. By seamlessly integrating speed, accuracy, and accessibility, this platform has the potential to transform liquid biopsy, bringing precision medicine to the forefront of widespread clinical practice.” said Jessica F. Liu.
Authors of the paper include Jessica F. Liu, Jianping Xia, Joseph Rich, Shuaiguo Zhao, Kaichun Yang, Brandon Lu, Ying Chen, Tiffany Wen Ye, and Tony Jun Huang.
This research was supported by the National Institutes of Health (grant nos. R01GM132603, R01GM141055, and R01GM135486), National Science Foundation (CMMI-2104295), and National Science Foundation Graduate Research Fellowship (2139754).
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