Lab-on-a-Chip & Microfluidics 2019: Emerging Themes, Technologies and Applications Track "A" Agenda

Conference Chaiperson Opening Remarks and Welcome to the Attendees
Leanna Levine, Founder & CEO, ALine, Inc., United States of America

Conference Chaiperson Opening Remarks and Welcome to the Attendees
Amy Adelberger, Founder and CEO, Global Impact Advisors, United States of America

Microfluidic Methods for Single-Cell Manipulation and -Omics Analyses
Aaron Wheeler, Canada Research Chair of Bioanalytical Chemistry, University of Toronto, Canada

Polymer-based Nanosensors for Single-Molecule Sequencing
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

We are generating a single-molecule DNA/RNA sequencing platform that can acquire sequencing information with high accuracy (>95%) at unprecedented throughputs (106 bases s-1). The technology employs high density arrays of nanosensors that read the identity of individual mononucleotides from their characteristic flight-time through a 2-dimensional (2D) nanochannel (~50 nm in width and depth; >10 µm in length) fabricated in a thermoplastic via nanoimprint lithography (NIL). The mononucleotides are generated from an intact DNA fragment using a highly processive exonuclease, which is covalently anchored to a plastic solid support contained within a bioreactor that sequentially feeds mononucleotides into the 2D nanochannel. The identity of the mononucleotides is deduced from a molecular-dependent flight-time through the 2D nanochannel. The flight time is read in a label-free fashion by measuring current transients (i.e., resistive pulse sensing) induced by a single mononucleotide when it travels through a constriction with molecular dimensions (<5 nm in effective diameter) poised at the input/output ends of the flight tube. In this presentation, our efforts on building these nanosensors using NIL in thermoplastics will be discussed and the detection of single molecules using electrical transduction with their identity deduced from the associated flight time provided. Also, surface modifications of thermoplastics for the immobilization of biologics, such as exonucleases, will be discussed as well as the activity of biological enzymes when immobilized to a plastic support. Finally, information on the manipulation of single DNA molecules using nanofluidic circuits will be presented that takes advantage of forming unique nanoscale features to shape electric fields for DNA manipulation and serves as the operational basis of the nanosensing platform.

Approaches to Wearable Microfluidic Sensor Devices for Well-Being and Personalized Healthcare
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

A new generation of portable, wearable biomedical devices is becoming possible through recent advances in microfluidic technologies, microelectronics, sensors and biosensors. Key to this integration is the phenomenal growth in mobile computing power provided by current tablets and mobile phones.  Through miniaturization and carefully engineered smart designs we can embed computer control and analytical best practice into portable even wearable devices that are able to compensate for shortcomings such as falling performance. These hybrid microfluidic systems appear to their target users as simple stable systems that tell them what they want to know. My group specializes in designing and building such microfluidic systems to meet the needs of acute critical care medicine. Key molecular markers are measured using both optical and electrochemical sensors and biosensors. We then work with clinical care teams to show proof of concept of the real-time continuous chemical information that microfluidic systems can produce. Our ultimate goal is that such systems can be used to monitor patients and guide therapy in a patient-specific, personalized way.

Rapid Diagnostics of Antibiotic Resistance
Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering, California Institute of Technology, United States of America

Rapid antibiotic susceptibility testing (AST) is critical for determining appropriate treatments and for enabling global antibiotic stewardship.  Phenotypic AST is the gold standard, but these tests are unacceptably slow, requiring pre-culture steps.  Genotypic AST methods are not sufficiently general to replace the gold standard (especially in Gram-negative organisms).  Our lab uses microfluidic digital single-molecule counting of pathogen-specific RNA and DNA to perform phenotypic AST directly from clinical samples in as fast as 30 min. Further, we have demonstrated refinements of this approach that can enable rapid AST in high-priority organisms such as Neisseria gonorrhoeae and Carbapenem-resistant Enterobacteriaceae (CRE).

Optical and Electrical Single Molecule Analysis With Nanopore Optofluidic Devices
Holger Schmidt, Narinder Kapany Professor of Electrical Engineering, University of California-Santa Cruz, United States of America

In recent years, single molecule analysis has gained increasing importance for next-generation medical diagnostics. Among the leading approaches are optical fluorescence detection and electrical analysis of ionic current across nanopores. I will discuss an integrated platform that can combine both approaches in a single chip. Design and applications of these electro-optofluidic chips will be discussed. These include multiplex detection of multiple molecular biomarkers, dual-mode detection of single viruses, and feedback controlled gating of a nanopore for on-demand delivery and selection of biomolecules for chip-based analysis. The advanced level of electrical, optical, and fluidic integration is ideal for point-of-care applications.

The Difficult First Step for POC Diagnostics
Paul Yager, Professor, Department of Bioengineering, University of Washington, United States of America

A diagnostic process, no matter how long, begins with a first step.  Whether to guide treatment of an individual’s infection, or to control the outbreak of a pandemic, there is an urgent need for low-cost rapid diagnostic devices capable of identifying the cause of infectious disease that work wherever the person is, not just in a centralized laboratory.  “Ubiquitous diagnostics” can bring the best diagnostic capabilities to homes, physicians’ office laboratories and pharmacies in the developed world, or to places in the developing world where nothing is available now.  However, the greatest challenge is often not detection of the analyte(s), but the preparation for the sample for that detection process.  Samples differ in volumes, the concentration of the analyte, and the presence of components that can interfere with analyte detection.  We have been working to develop simple devices for nucleic acid amplification tests (NAATs) for the presence of Chlamydia and gonorrhea, Mycobacterium tuberculosis and HIV.  To enable them we have created a suite of stand-alone or integrated sample-handling components that can process blood, urine or swabs from different cavities in the body: the goal in all cases is to create a few microliters of a fluid that is both concentrated and “clean” enough that it can be introduced automatically into a sensitive NAAT device of our own design.  We will show recent progress in reducing such processes to a few user-friendly steps appropriate for untrained users.

Cell-based Point-of-Care Oncology Tool (POCOT) For Precision Medicine
John McDevitt, Chair, Department Biomaterials, New York University College of Dentistry Bioengineering Institute, United States of America

The U.S. health care spending in 2016 reached $3.3 trillion or $10,348 per person. Health spending now accounts for 17.9% the US Gross Domestic Product. Chronic diseases like cardiac and cancer account for the majority of the costs in this area. Further, contributing to the $2.8B total costs for each new pharmaceutical is the cumbersome decade long approval process. These somber statistics seem to be offset by the promise of cutting edge biomarker developments with 157,000 biomarker papers published in last decade, yet only ~1 biomarker per year gets approved by the FDA. Likewise, the true potential for precision medicine is tampered by an infrastructure and incentive structure that slows the arrival of the benefits of precision medicine. To overcome these limitations, transformative new tools are desperately needed to enable a new era of medicine through research, technology, and policies that empower patients, researchers, and providers to work together toward development of individualized treatment. This talk features the development, optimization and validation of the first cell-based point-of-care oncology tool (POCOT) for precision medicine. Using single-cell data collected non-invasively from cytology samples of prospectively recruited patients with gold-standard-confirmed diagnoses, a series of predictive models were developed and validated resulting in a “continuous numerical risk score”. Model development consisted of: (1) training binary classification models for each diagnostic class pair, (2) pairwise coupling to obtain diagnostic class probabilities, and (3) a weighted aggregation to obtain a final risk score on a continuous scale.  Diagnostic accuracy based on optimized cutpoints for the validation dataset ranged from 76% for Benign lesions, to 82.4% for Dysplastic, 89.6% for Malignant, and 97.6% for Normal controls. The weighted aggregation of pairwise probabilities into a continuous variable was associated with a minor decrease of 6.4% for overall accuracy and demonstrated a strong positive relationship with diagnostic severity (Pearson’s coefficient = 0.805 for validation dataset). Frequencies of 5 cellular phenotypes recorded for 3 different ranges of the numeric index score agreed with common trends observed in conventional cytopathology. Finally, a simulation demonstrated that the numeric index can respond to changes in as few as 1% of the cells within a cytology sample, which is a necessary requirement for future evaluations of its utility for lesion monitoring.  The first cell-based point-of-care oncology tool for precision medicine is demonstared in the context of a continuous numeric index for potentially malignant oral disorders. These efforts result in a sensitive, accurate, and non-invasive method for enabling monitoring of these lesions. Continuous quantitative severity indices devoid of human subjectivity and categorization bias could have significant implications in a variety of medical decision making situations where microscopic evaluations and grading of biopsy samples by pathologists currently determine diagnostic, prognostic and treatment decisions. As such, this numeric index has the potential to monitor disease progression, recurrence, and the need for therapeutic intervention.

Enzyme-based Bioelectronic Wearable Sensing Devices
Joseph Wang, Distinguished Professor, SAIC Endowed Chair, University of California-San Diego, United States of America

Wearable bioelectronic devices rely on oxidoreductase enzymes and have already demonstrated considerable promise for on-body applications ranging from highly selective non-invasive biomarker monitoring to epidermal energy harvesting. Critical to such progress is the judicious design of the enzyme-electronic interface, along with flexible platforms with mechanical properties similar to those of biological tissues. Such devices require special attention to the enzyme-electronic interface and to several considerations related to wearable applications, such as mechanical properties (flexibility and stretchability), operational stability in different biofluids and under changing conditions (e.g., pH, temperature), biofouling, selectivity, and low target concentrations. Keeping these requirements in mind, our group has pioneered a variety of wearable biocatalytic sensors and biofuel cells devices. By leveraging the advantages of biocatalysis, electrochemistry, and flexible electronics, and addressing key challenges, wearable bioelectronic devices could have a tremendous impact on diverse biomedical, fitness and defense fields.

New Tools For Liquid Biopsy: Rare Cells, Exosomes, and Circulating DNA
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

This presentation will describe new technologies we developed for liquid biopsy and precision medicine.

Fast, Cartridge-Ready Sample Prep Using Off-the-Shelf Components
Richard Chasen Spero, CEO, Redbud Labs, United States of America

Sample-to-answer products are catching fire in the diagnostics and research markets, but microfluidic sample prep remains a major challenge. Methods that are ubiquitous at the bench, such as magnetic beads and centrifugation, translate poorly to cartridge formats. Meanwhile, microfluidic sample prep methods are generally proprietary and challenging to customize. We demonstrate microfluidic sample prep using commercially available components to deliver rapid affinity purification of target analytes. The result is an open platform for cartridge-ready sample prep that can deliver for lab-on-a-chip developers what magnetic beads enable at the bench.

Free-Surface Microfluidics and SERS or High Performance Sample Capture and Analysis
Carl Meinhart, Professor, University of California-Santa Barbara, United States of America

Nearly all microfluidic devices to date consist of some type of fully-enclosed microfluidic channel. The concept of ‘free-surface’ microfluidics has been pioneered at UCSB during the past several years, where at least one surface of the microchannel is exposed to the surrounding air.  Surface tension is a dominating force at the micron scale, which can be used to control effectively fluid motion. There are a number of distinct advantages to the free surface microfluidic architecture.  For example, the free surface provides a highly effective mechanism for capturing certain low-density vapor molecules.  This mechanism is a key component (in combination with surface-enhanced Raman spectroscopy, i.e. SERS) of a novel explosives vapor detection platform, which is capable of sub part-per-billion sensitivity with high specificity.

Circulatory System on a Chip -- From in vitro to in vivo, From Single Cell to Microphysiological Systems
Abraham Lee, Chancellor’s Professor, Biomedical Engineering & Director, Center for Advanced Design & Manufacturing of Integrated Microfluidics, University of California-Irvine, United States of America

The circulatory system is a critical physiological process of the human body that maintains homeostasis by balancing biological parameters by the delivery and removal of nutrients/waste and fighting off invading pathogens.  Through the advancement of microfluidics technologies, we have enabled the automation of biological fluids delivery through physiological vasculature networks that mimic the physiological circulation of the human body.  The critical bottleneck is to engineer the microenvironment for the formation of 3D tissues and organs and to also pump and perfuse the tissue vascular network for on-chip microcirculation.  On the other hand, microfluidics play an important role in the recent advances in liquid biopsy, an emerging technique that analyzes biological samples such as blood for the detection of biomolecules or cells that are indicative of disease or physiological state.  Specifically, liquid biopsy has become a promising technology to isolate and target rare cells such as circulating tumor cells (CTCs) in body fluids thanks to many of these microfluidic cell sorting techniques.  This advent of microfluidic liquid biopsy provides an in vitro snap shot into the patient’s physiological status via the in vivo circulation that enables one to monitor disease state and progression for diagnosis and prognosis. A key bottleneck is to identify the critical subpopulation of cells, often at single cell resolution among billions of cells in circulation. Along with the aforementioned in vitro on-chip perfused vascularized tissue platforms, these two technologies go hand-in-hand to connect in vitro screening to in vivo screening with great potential in the development of personalized medicine.  Ultimately this is the microfluidic maintenance of physiological equilibrium, or ‘microfluidic homeostasis.'

A Mobile Healthcare System Based on Smartphone and Smart Lab-on-a-Chip using Microchannel Capillary Flow Assay (MCFA)
Chong Ahn, Distinguished University Research Professor, Mitchell P. Kartalia Chair Professor of BioMEMS, University of Cincinnati, United States of America

A new mobile healthcare system using smartphone and smart lab-on-a-chip for the rapid and high sensitive detection of malaria biomarker, Plasmodium falciparum Histidine Rich Protein 2 (PfHRP2), has been developed and characterized in this work.  Chemiluminescence-based sandwich enzyme linked immunosorbent assay (ELISA) for PfHRP2 as the target antigen was performed on the lab-on-a-chip for high sensitive microchannel-based capillary flow assay (MCFA). A limit of detection (LoD) of 1.2 ng/ml of PfHRP2 was achieved, which is considered enough for the diagnostics of possible malaria infection.  A smartphone based self-powered portable analyzer was also developed for optical detection.   An optical detection system, which can interface with a smartphone, has been developed for the chemiluminescence detection.  The results obtained from this work envisages a new stand-alone mobile healthcare system for the rapid diagnosis of infectious diseases using a smartphone.

Droplet Emulsion Generation based on Electrokinetics: Digital PCR and Cell Encapsulation
Hsueh-Chia Chang, Bayer Professor of Chemical and Biomolecular Engineering, University of Notre Dame, Interim Chief Technology Officer, Aopia Biosciences, United States of America

We report a new microdroplet generation technology that uses AC electric field rather than hydrodynamic shear to generate droplets. Intricate flow-rate tuning of both phases is hence unnecessary and massive parallelization is now possible because of the absence of hydrodynamic cross-talk.   The bulky and expensive precision micropump is replaced by a simple and miniature AC source that can be driven by a battery.  With a less viscous oil phase and a properly tuned AC frequency, we are able to eliminate electro-coalescence and Rayleigh fission.  Monodispersed droplets with CV less than 3% and with a size range from 10 to 100 microns can be generated at a high throughput of nearly 1000 Hz per nozzle.  Moreover, the size of the droplets can be easily adjusted by simply tuning the voltage or the frequency.  One hundred to one million droplets and, with parallelization,  more than 1 billion droplets can be generated in 30 minutes.  This large range allows high-dynamic range digital PCR and conformal cell encapsulation.

Improving Sustainability in the Lab-On-a-Chip Field: Environmentally-Friendly Device Prototyping and Manufacturing
Maiwenn Kersaudy-Kerhoas, Professor of Microfluidic Engineering, Heriot-Watt University, United Kingdom

The use of single-use, disposable medical equipment has increased the amount of medical waste produced and the advent of point-of-care diagnostics in lab-on-chip format is likely to add further volume. Current materials used for the manufacture of these devices are derived from petroleum sources and are, therefore, unsustainable. In addition, disposal of contaminated plastics necessitates combustion to reduce infection risk, which has, depending on material composition, and incineration capacity, an undesirable environmental impact. To address these issues, we have developed several sustainable approaches for the rapid prototyping of single-use point-of-care cartridges, including use recycled PMMA or laminated PLA. Parameters such as optical quality, cost, and translation to mass-market products will be discussed. These techniques present exciting opportunities for immediate and future sustainable solutions in the LOAC field.

A Fully Automated Lab-on-a-Chip Platform For Biological Applications
Zeynep Altintas, Head of Biosensors and Receptor Development Group, Technical University of Berlin, Germany

Our custom-designed microfludic sensor provides rapid, sensitive and specific diagnostic methods. It is capable of detecting carcinogenic toxins, various bacteria species and their DNAs, and serves as a promising tool for pathogen detection in environmental and medical samples.

Quantum Diagnostics: From Single-Cells to Single-Molecules
Dino Di Carlo, Armond and Elena Hairapetian Chair in Engineering and Medicine, Professor and Vice Chair of Bioengineering, University of California-Los Angeles, United States of America

The ultimate limits of diagnostics in biology are the “quantum” units that convey information, e.g. single nucleic acids, proteins, and cells. Microfluidics has emerged as a powerful tool to compartmentalize single cells and molecules into sub-nanoliter droplets as individual bioreactors to enable sensitive detection and analysis down to this quantum limit. However, the current systems for quantum diagnostics have not been widely adopted, partly due to the requirement of specialized instruments and microfluidic chips to generate uniform droplets and perform adequate manipulations.  I will discuss the platforms we are developing to fractionate volumes in simplified, instrument-free ways using 3D-shaped microparticles. Each “lab-on-a-particle” can be analyzed using widely available flow cytometers. These new lab-on-a-particle reagents eliminate the need for specialized new equipment for microfluidic compartmentalization and readout and promise to democratize single-molecule and single-cell technologies.