Capture and label-free detection extracellular vesicles on gold-nanoisland based microfluidic Lab-on-a-CHIP device using syntheticpeptide Vn96

Background: Given the tremendous potential of circulating extracellular vesicles (EVs) for liquid-biopsy, there is great demand for simple, robust and clinically adaptable EV isolation and characterization Lab-on-aCHIP (LOC) platforms. Towards this, LOCs have been developed for capture, quantification and characterization of circulating EVs using EVsurface specific antibodies. The detection was performed either using fluorescent or label-free surface plasmon-resonance (SPR) sensors. The antibody-based isolation faces many challenges of quality control and shelf-life. To address the need for better affinity-based EV isolation method, we used a next generation affinity-based EV capture technology that uses a synthetic peptide (Vn96). Our group developed a LOC to capture EVs using Vn96, grafted onto gold nano-island (GNI) based on LSPR (localized SPR) sensing platform, and thus contributing to the emerging field of plasmofluidics. Methods: The LOC was built as: deposition of gold-nano-particle (GNP) on the glass surface and annealing of those deposited GNP to form GNI, bonding of PDMS onto the GNI and simultaneous LSPR in each spectrum. We have used scanning electron microscopy, atomic force microscopy, tunable resistive pulse sensing to count enriched EVs on LOC and relevant molecular analysis. Results: We designed, simulated and fabricated LOCs to identify the best microfluidic channel design on PDMS which were bonded on to a glass surface containing GNI grafted with Vn96-peptide using chemistry to covalently attach streptavidin onto the GNI followed by attachment biotinylated Vn96. At each steps of tagging streptavidin to affinity attachment of EV onto Vn96 was quantitated using LSPR to identify parameters for the best efficiency. Our results demonstrated that Vn96grafted LOC enriched EVs as a function of red-shift in the pick-LSPR spectra and was further characterized by eluting the attached EV from LOC for counting, imaging and molecular characterization. Summary/Conclusion: Our results demonstrate that Vn96-based affinity enrichment of EVs can be adapted on plasmofluidic platform using label-free quantification. We are advancing our current results to integrated LOC to perform complete hand-free protocol: from EV enrichment to multi-parametric molecular analysis.