The invention provides a "sample-in-answer-out" microfluidic exosome profiling platform which integrates immunoaffinity isolation of plasma exosomes with quantitative detection of both surface and intravesicular markers for characterization of exosomal phenotypes.
Tumor-derived exosomes have attracted increasing interest for non-invasive cancer diagnosis and monitoring of treatment response. However, biology and clinical unitization of exosomes remains low, due to current technical challenges in rapid isolation and molecular analysis of exosomes. This invention overcomes current technical limitations and delivers a new microfluidic platform to expedite the exosome isolation and molecular analysis pipeline into one single chip-sized device.
The multifunctional integration and automation presented in this invention confers new capability for a broad range of molecular assay of circulating vesicles, including probing of intravesicular microRNA, DNA, proteins, as well as the exosomal surface markers.
A cascading microfluidic circuit connected by sophistic channel network allows (1) Isolate circulating exosomes /microvesicles /membrane vesicles directly from human samples (e.g., blood, urine, saliva) and biological samples (e.g., cell culture media); (2) Probe and identify exosomal markers (e.g., proteins, microRNA, DNA); (3) Detect intravesicular markers and correlate with its surface marker for molecular classification and phenotyping of exosomes/vesicles.
In comparison to conventional bench-top methods, the microfluidic exosome analysis platform integrates sample preparation with molecular analysis into one automated pipeline, which decreases the manual intervention. The performance of this platform demonstrates increased assay sensitivity (LOD of femtogram), reduced assay time (minutes), and minimized samples and reagents consumption (uL). These advantages are beneficial for large-scale, comprehensive studies of exosomes at the systems level.
Compared to other microfluidic devices, it exhibits a three-fold distinction: 1) Offer a "sample-in-answer-out" capability for high-performance total analysis of exosomes by integrating cascading steps of isolation and molecular profiling in one microfluidic device; 2) Enable profiling of subpopulation landscapes in patient-derived exosomes defined by surface protein topography; and 3) Allow in-depth correlation of selectively isolated subpopulations with intravesicular markers, opening new ability to facilitate molecular definition of extracellular vesicles, and identification of biomarkers and therapeutic targets for cancer.
The platform invented here can be used in bioanalytical lab, molecular diagnostic lab, clinical test lab, biological and cancer research lab, hospital and clinical service.