Multiplex Liquid Biopsy Platform for Fractionation of Heterogeneous Vesicles and Precision Analyses of Their RNA Cargo
Hsueh-Chia Chang, Bayer Professor of Chemical and Biomolecular Engineering, University of Notre Dame, Interim Chief Technology Officer, Aopia Biosciences, United States of America

Extracellular RNAs in blood are promising circulating biomarkers.  However, as they can be easily degraded, extracellular RNAs must form complexes with proteins or are encapsulated by liposomes, exosomes or microvesicles to remain stable.  As a result, stable RNA biomarkers, like mRNA and miRNA, are carried by a heterogeneity of nanocarriers in blood with wide size, electrophoretic mobility and isoelectric point distributions.  Due to their different biogenesis, these carriers have specific surface proteins and are often selective in their choice of RNA cargoes.  To offer more precise quantification of the circulating RNA biomarkers, we have developed a suite of microfluidic modules that can fractionate the carriers by size, surface proteins and isoelectric point.  Size-based separation is achieved by an asymmetric nanoporous membrane whose conic pores reduce hydrodynamic resistance and vesicle fusion.  Extensive rinsing can be carried out to remove free-floating contaminating proteins like albumin.  We also developed several lysing modules that can lyse the vesicles and dissociate the complexes non-chemically, such that low-yield extraction can be eliminated as an intermediate step.  We integrate these modules with our membrane sensor, digital PCR module and nanopore sensor for RNA identification and quantification to achieve an integrated multiplexed platform for precise profiling of RNA cargoes in fractionated carriers.  Some of the upstream modules can also be integrated into a pretreatment platform for NGS.

Screening for Tumor-Derived Exosomes for Early Detection of Lung Cancer
Lydia Sohn, Almy C. Maynard and Agnes Offield Maynard Chair in Mechanical Engineering, University of California-Berkeley, United States of America

Late-stage diagnosis is the major contributor to the poor survival of lung cancer patients: 57% of patients diagnosed with lung cancer have metastatic disease and an abysmal 5-year survival rate of 5.7%.  The lack of symptoms specific to early-stage lung cancer and the low sensitivity and high cost of current screening methods (e.g. computed tomography) are current barriers to early diagnosis.  To address this challenge and enable lung-cancer screening, we are developing a sensitive, simple-to-use, and readily accessible platform to screen for lung tumor-derived extracellular vesicles (EVs) in saliva.  In this talk, I will discuss our platform, which is based on “Node-Pore Sensing” (NPS), and our work up to date in this challenging goal.

Cartridge-Ready Isolation and Concentration of Extracellular Vesicles
Richard Chasen Spero, CEO, Redbud Labs, United States of America

Despite decades of innovation, there is no accepted standard for isolation of circulating biomarkers in microfluidic systems. This creates a problem for developers of new biomarker assays and liquid biopsy tests. Traditional sample prep methods such as ultracentrifugation, affinity sorting with magnetic beads, or micro-filtration are acceptable for use in a discovery environment. However, these methods do not translate effectively to microfluidic cartridges, so promising assays must be completely re-implemented when moving from the bench to an integrated system. We present our work on a high-performance sample prep method for circulating biomarkers that translates easily from the bench to a microfluidic cartridge.

Near-Patient, Automated Platform for Rapid Microfluidic Extraction of Circulating Nucleic Acids from Milliliter Volumes of Whole Blood
Maiwenn Kersaudy-Kerhoas, Professor of Microfluidic Engineering, Heriot-Watt University, United Kingdom

Extracellular plasma circulating cell-free nucleic acids (CNAs) are promising clinical biomarkers but their measurement remains time-consuming, technically challenging and expensive. For CNAs to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue around CNAs stability in the presence of hemolysis, and few solutions for fast and robust extraction at the site of blood draw. We demonstrate a microfluidic system able to perform the extraction of circulating miRNAs from several milliliters of whole blood in a single disposable fluidic cartridge, on a fully automated platform, delivering a stable elution of CNAs in less than 45 minutes. The biological characterization of the eluates include qPCR, fluorometric and spectrophotometric analysis, and automated electrophoresis for fragment analysis. This platform enables the standardization of sample preparation at the point of blood draw and in resource limited settings and could aid the introduction of CNAs-based assays into routine clinical practice.

Nanostructures-Embedded Microchips for Liquid Biopsy in Cancer
Hsian-Rong Tseng, Professor, Crump Institute for Molecular Imaging, California NanoSystems Institute, University of California-Los Angeles, United States of America

The current gold standard for cancer diagnosis is the characterization of tumor tissues acquired via invasive procedures, e.g., surgical excision or needle biopsy. As an alternative to solid tumor biopsy, many have proposed the use of a “liquid biopsy” based on blood components like circulating tumor cells (CTCs) and extracellular vesicles (EVs). By detecting, enriching, and analyzing CTCs and EVs, we will be able to noninvasively and dynamically monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status. Over the past decade, our research team at UCLA pioneered a unique concept of “NanoVelcro” CTC Chips and “NanoVilli” EV Chips, in which CTC and EV capture agent-coated nanostructured substrates were utilized to immobilize CTCs and EVs with remarkable efficiency, respectively. Multiple generations of NanoVelcro CTC and NanoVilli EV Chips have been developed over the past decade for a variety of clinical utilities, e.g., noninvasive molecular analysis for monitoring disease progression and treatment intervention.  In this presentation, I will summarize the development of the new generations of “NanoVelcro” CTC and “NanoVilli” EV Chips, and the clinical applications of these new in vitro molecular diagnostic (IVMD) devices for cancer.

Extracellular Vesicles For Early Diagnosis of Stable Angina in a General Practitioner Environment
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Cardiovascular Disease (CVD) is with cardiovascular events of Ischemic Heart Disease (IHD) and Stroke, the number 1 and 2 cause of death in the world.

Stable Coronary Artery Disease (SCAD) or Stable Angina (SA) is one of the entities of IHD but in contrast to hsTroponins for myocardial infarction, there is no bloodmarker for quick and easy diagnosis.
SA is a very common disease (10-14% in the age group of 65-84 years) and a serious pathology resulting in a 4-6 times higher change of cardiovascular events like myocardial infarction. SA diagnosis is challenging, as many patients present with atypical symptoms with women have a different symptom sensation than men. This results in that 90% of women with chest pain suspected for SA are referred to the hospital by the GP do not have SA.
A simple SA early diagnosis blood test is expected to allow GPs to improve their assessment and decisons to refer or not to refer a patient to a cardiologist, reducing care costs and efficiency of care trajectories.
We have now a validated protein signature measured  in subsets of plasma extracellular vesicle (EV) that can accurately diagnose SA in women using 25 ul plasma. We simplified and validated this EV-based blood assay and will now develop a microfluidic device to isolate plasma EV subsets and determine EV biomarker levels that will make early diagnosis of SA in the GP environment possible.
Such a microfluidic device will also be very useful in other POC applications using EV subsets in diagnosis and prognosis of cardiovascular disease in GP or ambulance.