Abstract 994: Chiroptical detection and mutation analysis of cancer-associated extracellular vesicles in microfluidic devices with oriented chiral nanoparticles
Authors: Kang Yoon-Tae , Kim Ji-Young , Turali-Emre Emine Sumeyra , Jang Hee-Jeong , Cha Minjeong , Kumari Abha , Palacios-Rolston Colin , Subramanian Chitra , Purcell Emma , Owen Sarah , Lim Chung-Man , Reddy Rishindra , Jolly Shruthi , Ramnath Nithya , Kotov Nicholas A. , Nagrath Sunitha 
Published: April 2023

Abstract
Small extracellular vesicles, often termed as “exosomes” carry informative cargo containing proteins and lipids, reflective of their cellular origin. Thus, they are promising biomarkers for early diagnosis of cancer. However, conventional profiling methods like quantitative polymerase chain reaction (qPCR) require complex procedures, thereby limiting the analytical sensitivity of exosomes for liquid biopsies. Here, we demonstrate a sensitive microfluidic device (CDEXO) that isolates and profiles cancer-associated exosomes directly from blood plasma using assembled chiral gold nanoparticles (AuNPs). The unique changes in chiral signals are associated with specific biomolecules on the exosomes’ membranes. Thus, we can distinguish exosomes of lung cancer patients from those of healthy individuals and detect mutated EGFR proteins on the membrane. Hence, this low-cost microfluidic device is an attractive technique for rapid, sensitive, and versatile profiling of various extracellular vesicles.
Methods: The top layer of CDEXO devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The bottom glass slide was functionalized with a layer-by-layer assembly of cationic poly(dimethyl diallyl ammonium chloride) and anionic polystyrene sulfonate. AuNPs were prepared by adding gold nanoplates to a growth solution. Next, AuNPs were conjugated with biotinylated Annexin-V, using Neutravidin-biotin chemistry. Exosomes were harvested from lung cancer cell lines (A549, H1650, H3255) and lung fibroblasts (MRC5) and spiked into the CDEXO chip. EDTA was used to release the captured exosomes and quantified using nanoparticle tracking analysis (NTA). CD spectra were measured by spectrometry. Imaging of AuNPs and exosomes were done using scanning electron microscope (SEM).
Results: CDEXO captures cancer-associated exosomes with an efficiency of 81.1±1.5%. H3255 derived exosomes that exhibited EGFR point mutation showed the greatest change in spectral signals from the baseline, followed by A549 (wild type), H1650, (EGFR exon19 deletion) and MRC5 (healthy). The CD responses were measurable at exosome numbers as low as 100. Further validation with 19 lung cancer patients showed an average 40% change in chiral signals from isolated exosomes that were 5.6 times higher in patients than healthy donors.
Conclusions: A microfluidic device with chiral AuNPs allows sensitive and accurate detection of lung cancer-associated exosomes by conjugation with Annexin V. The resulting strong CD peaks that arise from specific interactions between exosomal surface proteins and chiral AuNPs facilitate in-depth profiling of target exosomes, including EGFR mutation expression.
Citation Format: Yoon-Tae Kang, Ji-Young Kim, Emine Sumeyra Turali-Emre, Hee-Jeong Jang, Minjeong Cha, Abha Kumari, Colin Palacios-Rolston, Chitra Subramanian, Emma Purcell, Sarah Owen, Chung-Man Lim, Rishindra Reddy, Shruthi Jolly, Nithya Ramnath, Nicholas A. Kotov, Sunitha Nagrath. Chiroptical detection and mutation analysis of cancer-associated extracellular vesicles in microfluidic devices with oriented chiral nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 994.