Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating

Abstract

Exosomes are cell-derived nano-sized vesicles that have been recently recognized as new mediators for many cellular processes and potential biomarkers for non-invasive disease diagnosis and the monitoring of treatment response. To better elucidate the biology and clinical value of exosomes, there is a pressing need for new analytical technologies capable of the efficient isolation and sensitive analysis of such small and molecularly diverse vesicles. Herein, we developed a microfluidic exosome analysis platform based on a new graphene oxide/polydopamine (GO/PDA) nano-interface. To the best of our best knowledge, we report for the first time, the GO-induced formation of a 3D nanoporous PDA surface coating enabled by the microfluidic layer-by-layer deposition of GO and PDA. It was demonstrated that this nanostructured GO/PDA interface greatly improves the efficiency of exosome immuno-capture, while at the same time effectively suppressing non-specific exosome adsorption. Based on this nano-interface, an ultrasensitive exosome ELISA assay was developed to afford a very low detection limit of 50 μL(-1) with a 4 log dynamic range, which is substantially better than the existing methods. As a proof of concept for clinical applications, we adapted this platform to discriminate ovarian cancer patients from healthy controls by the quantitative detection of exosomes directly from 2 μL plasma without sample processing. Thus, this platform could provide a useful tool to facilitate basic and clinical investigations of exosomes for non-invasive disease diagnosis and to aid precision treatment.

Figure 1

The nano-interfaced microfluidic exosome platform (nano-IMEX). (A) Schematic of a single-channel PDMS/glass device, with the exploded-view highlighting the coated PDMS chip containing an array of Y-shaped microposts. (B) Surface of the channel and microposts coated with graphene oxide (GO) and polydopamine (PDA) as a nanostructured interface for the sandwich ELISA of exosomes with enzymatic fluorescence signal amplification. (C) The procedure for surface functionalization of the microfluidic chips.

Figure 2

Characterization of microfluidic GO/PDA coating. (A) SEM image and digital image (inset) of a GO/PDA-coated chip containing the Y-shaped PDMS microposts. (B) SEM image of a GO-coated channel (inset) showing the microscale 3D surface topology formed by GO coating. (C, D) SEM of the GO/PDA-coated channels with different reaction time for PDA deposition (inset) showing distinct morphologies of the GO/PDA interface. (E) SEM image of the PDA-coated channel (inset) showing a much smoother and more solid PDA film formed on the surface without GO coating. (F) Raman spectra of the coatings. Inset: red shift of the G band of GO after PDA coating. The GO/PDA plot is offset for comparison.

Figure 3

Evaluation of the specificity of exosome capture by the GO/PDA chip. (A) SEM examination of non-specific exosome capture on a GO/PDA interface without an antibody. (B) SEM image showing densely captured COLO-1 cell exosomes on a GO/PDA surface coated with the CD81 antibody. Inset: cup-shaped morphology of the exosomes. (C) Comparison of exosome ELISA readout and non-specific background levels obtained with the chips coated by GO/PEG, GO/PDA and PDA only, respectively. The exosome concentration was 5 × 10⁴ µL⁻¹.

Figure 4

Characterization of the nano-IMEX chip using COLO-1 exosome standards. (A) Comparing the GO/PDA interfaced and silane-treated chips for quantitative exosome detection. (B) Surface protein profiling of COLO-1 cell exosomes (10⁶ µL⁻¹) captured by CD81 mAb.

Figure 5

Clinical analyses of plasma-borne exosomes in ovarian cancer (OvCa). (A) Calibration curves for detecting exosomes pre-purified and directly from patient plasma. (B) Boxplots overlaid with dot plots for clinical sample analysis by nano-IMEX chips. (C) Bradford assay of the total exosomal proteins and (D) NTA counting of exosomes purified from the same samples used in (B). (***, p < 0.001; **, p < 0.01). (E) Detection of plasma exosomes in an OvCa patient before and after treatment.