Recent discoveries have shown that microvesicles, called exosomes, often take on a pivotal role in cell-to-cell messaging, serving as the transport platform that carries messenger RNA (mRNA) and microRNA (miRNA). Crucially, this cell-to-cell communication can affect gene transcription and ultimately, the protein production of recipient cells, often converting them to a disease state. For this reason, exosomal RNA is believed to play an important role in a wide variety of biological functions, such as adaptive immune response and cardiac tissue repair, as well as in the formation and development of diseases ranging from Alzheimer's disease and multiple sclerosis to cardiovascular disease and cancer. Further, exosomes are found in most biological fluids, including blood, saliva, and urine, and therefore are prime to serve as biomarkers for early disease detection. However, extracting exosomes and analyzing the RNA they carry, is a long, involved, and expensive experimental task, requiring multiple steps of separation, isolation, and manipulation. In this research program, we will develop a low-cost, high-throughput microfluidic platform that rapidly isolates exosomes from biological samples and sensitively detects the mRNA and miRNA they carry. We envision that this novel instrument for by detecting and quantifying exosomal RNA will have a major impact on how clinicians are able to diagnose early-stage disease and establish prognoses for disease development, becoming a game-changing diagnostic and therapeutic instrument. We will engineer the integrated device to be rapid, sensitive, and cost effective, requiring little biological material and utilizing disposable components. We will demonstrate the analytical proficiency of the microfluidic biochip by screening for a target biomarker exosomal miRNA in both healthy and cancerous pancreatic cell lines and establish the device's diagnostic and prognostic capability. The proposed microfluidic platform will separate and isolate exosomes from their background biological material and then subsequently lyse them and directly detect released target miRNA. We will utilize a size-exclusion membrane to separate the exosomes (~30-100 nm) from other biomolecules in complex samples (cell media, blood, ascites fluid). Surface acoustic waves (SAWs) generated on the surface of a piezoelectric microfluidic substrate will produce both high shear forces (via acoustic streaming) and high electric fields (via electro-mechanical coupling) to lyse the separated exosomes and release their RNA. We will detect miRNA with an ion-exchange membrane where immobilized oligonucleotide probes will hybridize with target miRNA and fundamentally alter the current-voltage relationship of the membrane. Our diverse, interdisciplinary research team will optimize and integrate each of these components, characterizing performance on a model system of exosomes secreted by epithelial cell lines from both healthy patients and those with pancreatic ductal adenocarcinoma, benchmarking against standard analysis techniques. Our goal is to reduce the analysis time from many hours/days using current methods to under 60 min with sub-pM limits of detection.

Public Health Relevance

This research will develop a microfluidics platform that can rapidly separate and isolate exosomes and detect and identify the microRNA and messenger RNA they carry. The projected speed and sensitivity of this device will enable the rapid and early detection of exosomal RNA that are biomarkers for conditions ranging from Alzheimer's disease and multiple sclerosis to cardiovascular disease and cancer, leading to diagnostic and prognostic advances in the fight against these diseases.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Exploratory/Developmental Grants (R21)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Smith, Michael
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University of Notre Dame
Engineering (All Types)
Biomed Engr/Col Engr/Engr Sta
Notre Dame
United States
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Marczak, Steven; Richards, Katherine; Ramshani, Zeinab et al. (2018) Simultaneous isolation and preconcentration of exosomes by ion concentration polarization. Electrophoresis :
Go, David B; Atashbar, Masood Z; Ramshani, Zeinab et al. (2017) Surface acoustic wave devices for chemical sensing and microfluidics: A review and perspective. Anal Methods 9:4112-4134