YLBT01: Late Breaking Poster Session Methodology Chairs: Muthuvel Jayachandran; Theresa Whiteside Location: Exhibit HallLBT01.Single vesicle counting enabled by DNA nanostructures Wenwan Zhong1; MMP-8 Proteins web Kaizhu Guo2; Wen Shen17:15 – 18:University of California, Riverside, Riverside, USA; 2University, Riverside, USABackground: Extracellular vesicles (EVs) may be valuable for sensitive and precise cancer diagnosis and prognosis, but their identification needs detailed molecular analysis in the EVs from various sources. Solutions: Single vesicle counting can overcome the noise limitation in batch evaluation and reveal the presence with the EVs carrying unique molecular signatures very indicative to their distinct cell of origin. Herein, we propose a very simple strategy to allow single vesicle counting and detect many exosome cargos in person vesicles. Our central hypothesis is that DNA nanostructures is usually established upon recognition from the molecular signatures on exosomes, and enable single EV counting and EV cargo profiling. Benefits: We’ve proved that DNA nanostructure (DNS) is often grown on exosome surface and enable detection of single vesicles employing conventional microscope or flow cytometer. DNS is established by Hybridization Chain Reaction (HCR) upon recognition of CD63. An initiator that consists of the aptamer sequence for CD63 and also a stem-loop structure was created so that binding to CD63 opened the stem for hybridization with Hairpin 1 (H1) and initiated the development of a lengthy dsDNA by way of continuous hybridization in between H1 and Hairpin 2 (H2). Only CD63 or MMP-28 Proteins site exosomes could initiate growth of long DNA merchandise from HCR as proved by gel electrophoresis. TEM also detected particles 500 nm in diameter just after the reaction, and also the mode diameter of the vesicles detected by Nanosight NS300 elevated by 50 nm. DNS enabled detection of exosomes inside the standard flow cytometer, whilst exosomes labelled with anti-CD63-conjugated QDs were not observed. Extra interestingly, the EVs carrying both CD63 and HER2 on its surface might be recognized by dual-labelling employing two initiators. The exosomes created by the breast cancer cell carry higher content of HER2 and CD63, but those from the non-tumour cell line MCF-10A exhibit low HER2 and high CD63 expression. When these exosome populations had been mixed at a two (SKBR3):1 (MCF-10A) ratio (particle concentration measured by NTA ahead of mixing), dual TIC-DNS could clearly differentiate the presence of two groups of exosomes. Summary/Conclusion: We believe our approach will help with identification of exosomes in clinical setting rapidly with low sample consumption. Funding: This study was funded by NIH R01CA188991.Methods: We propose EVs production in stirred tank bioreactor pursued by the tangential flow filtration (TFF) method (one hundred KDa cut-off cassette membranes) to purify the EVs. Wild sort EVs made by HEK293T cells had been cultured in suspension and on Corning enhanced attachment, Cytodex 1 and Cytodex 3 microcarriers and have been purified by ultracentrifugation or TFF. The bioreactor experiments have been performed in an Eppendorf BioFlo320 in 1 and three l vessels equipped with a pitched blade impeller. The culture inoculums were grown and expanded in T25, T75 and then, spinner flasks. Cytodex 1 microcarriers have been used to develop HEK 293T adherent cells. The suspension experiments had been performed in serum free medium (SFM II), Glutamax 1X, 8 CO2 and 37 , and for adherent cells five exosome depleted DMEM, 5 CO2 and.