Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip

Abstract

The performance of current microfluidic methods for exosome detection is constrained by boundary conditions, as well as fundamental limits to microscale mass transfer and interfacial exosome binding. Here, we show that a microfluidic chip designed with self-assembled three-dimensional herringbone nanopatterns can detect low levels of tumour-associated exosomes in plasma (10 exosomes μl^-1, or approximately 200 vesicles per 20 μl of spiked sample) that would otherwise be undetectable by standard microfluidic systems for biosensing. The nanopatterns promote microscale mass transfer, increase surface area and probe density to enhance the efficiency and speed of exosome binding, and permit drainage of the boundary fluid to reduce near-surface hydrodynamic resistance, thus promoting particle-surface interactions for exosome binding. We used the device for the detection-in 2 μl plasma samples from 20 ovarian cancer patients and 10 age-matched controls-of exosome subpopulations expressing CD24, epithelial cell adhesion molecule and folate receptor alpha proteins, and suggest exosomal folate receptor alpha as a potential biomarker for early detection and progression monitoring of ovarian cancer. The nanolithography-free nanopatterned device should facilitate the use of liquid biopsies for cancer diagnosis.

Fig. 1.. Multiscale Integration by Designed Self-assembly (MINDS).

(a) Conceptual illustration of the MINDS strategy that improves biosensing by 3D nanostructuring of microfluidic elements, e.g., herringbone (HB) mixer. Conventional solid HB mixer creates microvortices to promote mass transfer of targets. A particle will experience hydrodynamic resistance near a solid surface that reduces direct surface contact. In a 3D nanoporous HB (nano-HB) chip, fluid near the surface can be drained through the porous structure (red dashed lines) to increase the probability of particle-surface collisions. (b) The workflow for fabricating a 3D nano-HB chip by MINDS. (c) A nano-HB chip fabricated with 960-nm silica colloids. The digital photo exhibits Bragg diffraction of light (scale bar, 5 mm). The SEM images show a high-quality, crack-free HB array patterned on a glass substrate with a crystalline nanoporous structure. (d) Silica necks were formed between the contacting particles by treatment with 5% 3-MPS. (e, f) SEM images of mono-assembled nano-HBs with silica colloids of 520 and 170 nm. (g) A well-ordered binary lattice composed of 170 and 960 nm silica colloids at equal mass ratio. (h) A randomly organized nano-HB co-assembled with 520 and 960 nm silica colloids at a 1:1 mass ratio. (i) An anisotropic microstructure assembled from silica nanorods with an averaged diameter of 238 ± 32 nm and length of 1.34 ± 0.26 μm. (j, k) Fabrication of crack-free, 3D nanostructured sinusoidal ribbon and concave diamond arrays using designed microfluidic channels to engineer evaporative self-assembly of 960-nm colloids.

Fig. 2.. Fluidic characterization of nano-HB chips.

(a) Simulation of mixing two streams of 50-nm nanoparticles (NPs) and water co-flowing at 1 μL/min in a flat-channel, solid-HB, or nano-HB device, respectively. Zoom-in view over the first two HB units was shown. Scale bar, 400 μm. (b, c) Simulation results showing the transverse flow profiles across the channel width (b) and the streamwise flow profiles along the channel length (c), respectively. Insets highlight the flow patterns inside the nanoporous domains. Color contours indicate the velocity magnitude, while vectors represent the flow direction. The flow rate was 0.5 μL/min. (d) Fluorescence images (left) and transverse intensity plots (right) of mixing two flows of 46-nm fluorescent NPs and PBS injected at 0.25 μL/min in parallel. The intensity plots were measured along the channel width at three positions on the images as indicated. (e, f) Time-lapse plots of fluorescence intensity measured in the HB structures and the open grooves when a solution of 46 nm fluorescent NPs was pumped through a nano- or solid-HB chip at 0.5 μL/min, respectively. 3D confocal fluorescence microscopy images (left) were acquired when the channels were filled with the NP solution. (g) Time-lapse monitoring of diffusion of 46-nm NPs from the grooves into nano-HB structures. Flow pumping was stopped when the NP solution entered the observation region. The fluorescence image (left) was acquired at t = 60 min. (h) Representative 2D confocal microscopic images of the nano-HB (top) and solid-HB structures (bottom), respectively, with the NP solution flowing through the channels. The images were acquired at approximately 15 μm below the HB surface. Partial surface plots of the images (right) were displayed for the areas indicated by the dashed rectangles. Scale bar, 100 μm. In (e-h), nano-HB chips were assembled with 960 nm silica colloids. a.u., arbitrary unit. Error bars, one standard deviation (S.D., n = 3).

Fig. 3.. 3D engineered Nano-HB chip affords efficient immunocapture of exosomes.

(a) SEM images showing minimal non-specific absorption and immunocapture of COLO-1 exosomes (10⁵ μL⁻¹) on a non-modified (left) or a mAb-coated device (right), respectively. (b) Typical sphere- or cup-shaped morphologies and the clusters of COLO-1 exosomes captured on nano-HBs. Scale bars: 100 nm. (c) Representative size distribution of nano-HB-captured COLO-1 exosomes (n > 300) measured by SEM, compared to that of NTA analysis of UC-isolated vesicles. (d) 3D confocal fluorescence microscopy showing exosomes captured inside the nano-HBs. DiO-stained COLO-1 cell-derived exosomes were spiked in human plasma (10⁵ μL⁻¹). (e) Immunological and non-specific capture of COLO-1 exosomes as a function of injected sample volume assessed using an mAb or BSA-coated nano-HB chip, respectively. DiO dye-stained exosomes were spiked in PBS or 10× diluted human plasma (10⁶ μL⁻¹) and injected continuously through the chips at 0.5 μL/min. 2 μL eluent was collected at the outlet every 15 min and measured by a micro-volume plate reader to determine the reduction in fluorescence signal with respect to that of the original sample. (f) Comparison of standard UC isolation and the nano-HB capture of fluorescently stained exosomes of various cancer cell lines spiked in healthy plasma (10⁶ μL⁻¹). Signal decrease caused by exosome immunocapture was measured as in (e) and subtracted by that of non-specific adsorption to calculate the percentage of the corrected signal reduction, i.e., the specific capture efficiency. (g) Capture efficiency of nano-HB chip measured by the GAPDH mRNA level. EVs isolated and concentrated from SKOV3 and OVCAR3 cell culture media by UC were spiked in PBS (10⁶ μL⁻¹) and 100 μL of the solution was run on the chip. Captured exosomes were eluted out for droplet digital PCR (ddPCR) quantification. The GAPDH mRNA levels in chip-captured exosomes were normalized by that in the same amount of EVs measured without chip capture. (h) Profiles of six mRNA markers measured in the chip-captured SKOV3 and OVCAR3 exosomes in comparison with the vesicles isolated and concentrated by UC. Exosome capture and elution on the nano-HB chip was performed as in (g) and the levels of individual mRNAs measured by ddPCR were normalized against GAPDH. Anti-CD81 mAb was used for exosome capture in all cases. Error bars, one S.D. (n = 3).

Fig. 4.. Ultrasensitive detection of exosomes with the nano-HB chip.

(a) Engineering nano-HBs by the MINDS programs the sensitivity for detecting COLO-1 exosomes (10⁵ μL⁻¹). Statistic comparison between the 960 and 960/520 nm chips yielded p = 0.005. (b) Calibration curves for quantifying total exosomes by the flat-channel, solid-HB, and nano-HB chips. Serial 10× diluted COLO-1 exosome standards were used with a mixture of anti-CD9, CD63, and EpCAM mAbs for detection. (c) Western blot analysis of the protein markers in SKOV3 and OVCAR3 EVs with 10 μg BSA as negative control (NC). (d) Comparing the nano-HB chip and a standard microplate kit for ELISA detection of six proteins in SKOV3 and OVCAR3 exosomes. 20 μL, 10⁵ μL⁻¹ purified EVs were used for nano-HB assay, and 100 μL, 10⁶ μL⁻¹ EVs for microplate ELISA. All analyses were normalized against CD24 that was found most abundant. (e) The measurements of six targets in SKOV3 and OVCAR3 exosomes by the nano-HB chip and microplate-based ELISA correlated well. (f) Calibration curves for detecting the FRα+ subtype in UC-purified SKOV3 EVs spiked in PBS and a 10× diluted healthy plasma without detectable FRα. Total exosomes were captured with anti-CD81 mAb and the FRα+ subtype was detected with anti-FRα mAb. (g) High sensitivity of the nano-HB chip enables detection of FRα+ exosomes in an OvCa plasma sample, which is indiscernible to the conventional flat-channel chip. Two-tailed Student’s t-test was used at a significance level of p < 0.05. NS, not significant. (h) Protein profiling of exosomes directly in plasma from a control and two OvCa patients with nano-HB chips. Total exosomes were detected with a mix of CD9, CD63, and CD81 Abs. CD81 mAb was used for exosome capture in all cases. Error bars: one S.D. (n = 3).

Fig. 5.. Clinical profiling of circulating exosomes for diagnosis of ovarian cancer.

(a) Quantification of the exosomal levels of CD24, EpCAM, and FRα proteins directly from the plasma of OvCa patients (n = 20) and non-cancer controls (n = 10). Signals were subtracted by the corresponding background measursed with PBS to determine the protein levels. Error bars indicate S.D. (n = 3). (b) Comparison of the exosomal marker patterns measured with the nano-HB ELISA, the microplate ELISA, and the combined nano-HB capture and ddPCR of mRNAs. A subset of OvCa (n = 10) and control samples (n = 5) from (a) was assayed in triplicate and the mean values were normalized against CD24 in Patient #8 that showed the highest levels. For mRNA analysis, 100 μL plasma from each patient was diluted by 10 times and run through two 8-channel nano-HB chips to ensure fast and efficient exosome capture. (c) Typical images of immunofluorescence histological assays of the patient-matched tumor tissues (Patient #11). Scale bar, 50 μm. (d) Scatter dot plots of the plasma levels of three exosomal markers measured by the nano-HB assay for the patients of variable stages: control, early-stage (stage I/II), and advanced patients (stage III/IV). Error bars are the mean and one standard error of the mean (s.e.m.). Statistical comparison of three groups was performed by one-way ANOVA with post hoc Tukey’s test. Significance level was set at p < 0.05. (e) Heatmap constructed by non-supervised hierarchical clustering of the levels of exosomal CD24, EpCAM, and FRα recognizes the OvCa and control groups. Clustering analysis was performed with Ward linkage and Euclidean distance.