(683c) Rotational Brownian Motion of Immuno-Janus Particles Enables Rapid Quantification of Cancer-Associated Exosomes in Blood Plasma for Early Cancer Screening

Cancer is leading cause of death worldwide. Approximately 50% of cancers are diagnosed in advanced stages and 90% of cancer deaths are caused by metastasis. Early diagnosis facilitates more effective cancer treatments and improves patient quality of life, with many cancers exhibiting 5-year survival rates exceeding 80%. Many reports have shown that exosomes, small lipid-bound extracellular vesicles, hold significant potential for non-invasive cancer detection, even in early stages. With their dissociated and activated conformations for cancer-related binding, the proteins supported on the exosome lipid bilayer are much more relevant cancer markers than their dispersed counterparts. Yet, current exosome diagnostics are not without their drawbacks. A major limitation of exosome diagnostics is the extensive pre-treatment necessary to remove any non-specific binding agents, commonly involving hours to days of ultracentrifugation per sample. Once exosomes are isolated, the technologies used for characterization are often costly, not diagnostically pertinent, require lengthy incubation/washing steps, or use specialized equipment and training. Creating a rapid, easy to use technology that eliminates the need for pre-treatment steps is crucial for the effective utilization of exosomes as a diagnostic tool.

Our lab has developed a novel platform for directly quantifying plasma exosomes by measuring shifts in rotational Brownian motion of Immuno-Janus Particles (IJPs). These one-micron fluorescent polystyrene beads, coated with a thin 30 nm layer of gold on a singular hemisphere, exhibit a blinking phenomenon and offer rapid thiol-based antibody conjugation potential. Fundamental rotational Brownian motion induces a spin on the IJPs with a frequency inversely proportional to the cubed particle diameter. An increase in the effective size, i.e. via exosome coupling, decreases the speed with which the particles blink. Our specific and sensitive platform has shown a limit of detection of 1000 exosomes/μL using less than 10 μL of plasma. The size-based detection of IJPs circumvents the need for pre-treatment, as free-floating and non-specific proteins are too small to produce a signal. Implementing a custom single particle tracking algorithm allows for rapid (<60 minute) workflow for detection of exosomes.

Building upon this foundation, IJPs were functionalized with antibodies specific to cancer-associated surface markers such as aEGFR, CEA, and GPC1. We incubated the functionalized IJPs with plasma from cancer patients, including those with colorectal cancer (CRC), glioblastoma (GBM), and pancreatic ductal adenocarcinoma (PDAC). In a blind study encompassing over 90 clinical samples, we achieved differentiation between healthy and cancer patient plasma with an AUC of 0.92. Additionally, we successfully distinguished the types of cancers studied, achieving AUC values of 0.95 for CRC, 0.95 for GBM, and 0.92 for PDAC.