Streamlining sample preparation with high throughput SpinEx (Separation processing integration for Extracellular vesicles) - Extracellular vesicles (EVs) have emerged as a promising analyte to gain molecular insights into tumors through minimally invasive and repeated liquid biopsy. EVs carry a repertoire of biomolecules (e.g., proteins, nucleic acids) from their parent cells. Analyzing these markers offers a unique window into the dynamic status of tumors. Roadblock to clinical EV assays. Translating EVs into reliable clinical tests faces a significant technical hurdle: sample preparation. EVs exist within highly heterogeneous matrices, necessitating extensive sample purification for accurate downstream analyses. This need is particularly pronounced in blood, the predominant specimen type. Blood plasma contains various nanoscale vesicles, with lipoprotein particles (LPPs) outnumbering EVs by over a million times. Compounding this challenge is the similarity in sizes and densities between LPPs and EVs, making EV purification a formidable task. Conventional laboratory methods (e.g., ultracentrifugation, size-exclusion chromatography) are time-consuming and susceptible to co-isolating abundant LPPs with EVs. Although new microfluidic devices show promise in enhancing EV separation purity, their complexity and limited throughput have restricted widespread adoption in clinics. Innovations. This project aims to overcome these challenges by advancing a SpinEx (Separation-processing integration for Extracellular vesicles) technology. SpinEx is built on two core concepts: dual-mode chromatography (DMC) and disc fluidics. i) DMC leverages size and surface charge properties to selectively enrich EVs from plasma while eliminating LPPs. ii) Disc-based fluidics facilitate rapid, high-throughput flow operations with adaptable flow control by regulating disc spinning. Our pilot study successfully implemented DMC in a disc device and demonstrated efficient EV isolation. These breakthroughs inspire us to develop a fully automated, multifunctional SpinEx platform. In Aim 1, we will implement a SpinEx disc and a spinner system. We will design interchangeable fluidic modules for the disc unit, each dedicated to a specific task (EV isolation, protein labeling, RNA extraction). Users can customize workflows by plugging and playing these modules onto a disc frame. The spinner system will integrate a speed-controlled motor to rotate a disc and a mechanical arm designed to actuate fluidic valves within a disc. The developed SpinEx will undergo rigorous performance evaluation (e.g., EV purity, marker expression, reproducibility) and comparison with standard laboratory methods. In Aim 2, we will apply SpinEx to process clinical plasma samples for ovarian cancer diagnostics. SpinEx will isolate EVs and fluorescently label them for target protein detection. This aim will assess SpinEx's compatibility with real-world clinical samples. Impact. SpinEx's capabilities in rapid EV enrichment, high throughput, and modular functionality offer transformative potential in establishing robust clinical EV assays. This advancement could empower large-scale clinical studies on cancer biomarkers and democratize EV testing for improved early cancer detection and equitable care delivery.
