Lab-on-a-Chip and Microfluidics: Emerging Themes, Technologies and Applications Agenda

The Personalized Health Care Environment: How in vitro Diagnostics, Mobile Health and Medical Devices will Converge to Improve Health Care
Alan Wright, Chief Medical Officer, Roche Diagnostics Corporation, United States of America

Digital Microfluidics: A Platform Whose Time Has Come
Aaron Wheeler, Canada Research Chair of Bioanalytical Chemistry, University of Toronto, Canada

Digital microfluidics is a fluid-handling technique in which droplets are manipulated by electrostatic forces on an array of electrodes coated with a hydrophobic insulator. In this talk, I will present recent results from my group’s work with digital microfluidics, with an emphasis on the topics covered in this unique venue. Specifically, I will demonstrate how digital microfluidics is particularly well-suited for Lab on a Chip applications, given its ability to automate diverse laboratory processes on a generic, programmable platform. Likewise, I will report on our work using digital microfluidics for Point of Care Diagnostics and Global Health, reporting on the results of a field trial for measles and rubella diagnostics at refugee sites in Kenya. Finally, I will describe how digital microfluidics is emerging as a useful tool for Single-Cell Analysis and for integration with Mass Spectrometry to answer questions about cell heterogeneity and cell-cell communication. Through these examples, I will make the case that digital microfluidics is emerging as a useful new tool for the next generation of analytical techniques, across a wide range of applications.

Microfluidics and Sensors: New Tools for Real-Time Clinical Monitoring
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

A goal for modern medicine is to protect vulnerable tissue by monitoring the patterns of changing physical,  electrical and chemical changes taking place in tissue - ‘multimodal monitoring’. Clinicians hope such information will allows treatments to be guided and ultimately controlled based on the measured signals. Microfluidic lab-on-chip devices coupled to tissue sampling using microdialysis provide an important new way for measuring real-time chemical changes as the low volume flow rates  of microdialysis probes are ideally matched to the length scales of microfluidic devices. Concentrations of key biomarker molecules can then be determined continuously using either optically or electrochemically (using amperometric, and potentiometic sensors).  Wireless devices allow analysis to take place close to the patient. Droplet-based microfluidics, by digitizing the dialysis stream into discrete low volume samples, both minimizes dispersion allowing very rapid concentration changes to be measured, and allows rapid transport of samples between patient and analysis chip. This talk will overview successful design, optimization, automatic-calibration and use of both continuous flow and droplet-based microfluidic analysis systems for real-time clinical monitoring, using clinical examples from our recent work.

High-Performance Rapid Diagnostic Tests
Bernhard Weigl, Director, Center for In-Vitro Diagnostics, Intellectual Ventures/Global Good-Bill Gates Venture Fund, United States of America

Lateral flow and similar rapid diagnostic assays (LFAs) are easy to use and manufacture, low cost, rapid, require little or no equipment to operate, and do not need to be refrigerated. However, they are generally not considered to be very sensitive or able to provide a quantitative result. Our group believes that this lack of sensitivity is not a fundamental property of LFAs but rather a consequence of the way they are developed, manufactured, and marketed. Historically, most lateral flow tests were developed and optimized by relatively small manufacturers with limited R&D capabilities and budgets, and were generally used only for analytical targets prevalent at high concentration in patient’s samples that were relatively easy to measure. In contrast, our group’s mission is to develop LFA-based assays for use in global health applications that are as sensitive as the best conventional diagnostic assays (in some cases even better) while retaining all their cost, simplicity, and usability advantages.

Technologies for Personalizing Cancer Immunotherapies
James Heath, Elizabeth W. Gilloon Professor of Chemistry, California Institute of Technology (CalTech), United States of America

Cancer immunotherapy, which has taken virtually all aspects of oncology by storm over the past few years, is based upon using  cellular or molecular therapies to promote tumor cell/immune cell interactions.  At the heart of this therapy are the T cells that actually do the tumor cell killing, and the tumor antigens that are recognized by those T cells.  Recent work has shown that neoantigens play critical roles in many immunotherapy successes.  Neoantigens are tumor antigens that are fragments of mutated proteins expressed by the cancer cells, and contain those point mutations.  They are presented in the clefts of major histocompatibility molecules (MHCs) by many of the cells in the tumor, where they may be recognized by neoantigen-specific T cell populations.  In this presentation, I will discuss how various micro and nanotechnologies are being harnessed to identify, for a given patient which neoantigens are actively recruiting T cells into the tumor, and to carry out a deep molecular analysis of those neoantigen-specific T cells.  I will further discuss how that information can then be harnessed for personalized cancer immunotherapies in the form of neoantigen-based vaccines, or engineered T cell receptor adoptive cell transfer therapies.

Wearable Eccrine Sweat Biosensing: Uncovering The Real Challenges That Lie Ahead
Jason Heikenfeld, Professor and VP Operations, UC Office of Innovation, University of Cincinnati, United States of America

Despite the many ergonomic advantages of eccrine perspiration (sweat) compared to other biofluids (particularly in “wearable” devices),  sweat remains an underrepresented source of biomarker analytes compared to the established biofluids blood, urine, and saliva. Upon closer comparison to other non-invasive biofluids, the advantages may even extend beyond ergonomics: sweat might provide superior analyte information.  A number of challenges, however, have historically kept sweat from its place in the pantheon of clinical samples. These challenges include very low sample volumes (nL to µL), unknown concentration due to evaporation, filtration and dilution of large analytes, mixing of old and new sweat, and the potential for contamination from the skin surface.  More recently, rapid progress in “wearable” sweat sampling and sensing devices has resolved several of the historical challenges.  However, this recent progress has also been limited to high concentration analytes (µM to mM) sampled at high sweat rates (>1 nL/min/gland, e.g. athletics).  Progress will be much more challenging as sweat biosensing moves towards use with sedentary users (low sweat rates or not sweating at all) and/or towards low concentration analytes (pM to nM). Fortunately, none of the remaining challenges appear to be fundamentally blocking, and scientific and engineering innovations have the opportunity to enable broader application of sweat biosensing technology.

The Challenge in Building Phenotype Body-on-a-Chip Models for Toxicological and Efficacy Evaluations in Drug Discovery as well as Precision Medicine
James Hickman, Professor, Nanoscience Technology, Chemistry, Biomolecular Science and Electrical Engineering, University of Central Florida; Chief Scientist, Hesperos, United States of America

The utilization of human-on-a-chip or body-on-a-chip systems for toxicology and efficacy that ultimately should lead to personalized, precision medicine has been a topic that has received much attention recently. Key characteristic needed for these systems are the ability for organ-to-organ communication in a serum-free recirculating medium and incorporation of induced pluripotent stem cells that allow for understanding genetic variation as well as to construct systems utilizing stem cells from diseased patients and also from individuals. Additional characteristics that have been discussed are functional readouts that would enable non-invasive monitoring of organ health and viability for chronic studies that now are only possible in animals or humans at this time. In addition, in order to achieve wide spread adoption of these technologies they should also be low cost, easy to use and reconfigurable to allow flexibility for platforms to be examined with small variation. Our group, in collaboration with Dr. Michael Shuler from Cornell University, has been constructing these systems with up to 6 organs and have demonstrated long-term (>28 days) evaluation of drugs and compounds, that have shown similar response to results seen from clinical data or reports in the literature. We have accomplished the construction of these systems utilizing mostly 2D systems in serum-free medium with functional readouts that employs a pumpless platform that enables ease of use of these assays. Our group’s ability to control the interface between the biological and non-biological components in these systems has enabled the straightforward integration of multiple cell types in the same platform. Results with the functional multi-organ systems will be presented as well as results of five workshops held at NIH to explore what is needed for validation and qualification of these systems by the FDA and EMA.

Centrifugal Microfluidics for Biomedical Applications
Yoon-Kyoung Cho, Professor, Biomedical Engineering, Ulsan National Institute of Science & Technology; Group leader, IBS; FRSC, Fellow of Royal Society of Chemistry, Korea South

In this presentation, we will discuss our on-going research on “Lab-on-a-disc”, which applies centrifugal force to pump fluid for biochemical analysis. It is advantageous because of the capability to integrate and automate the analysis protocols into a disc-shaped device with simple, size-reduced, and cost-efficient instrumentation. We report various examples of fully integrated "lab-on-a-disc" for biomedical applications such as pathogen specific DNA extraction to test infectious diseases, multiplex enzyme-linked immunosorbent assay (ELISA), and isolation and analysis of circulating tumor cells starting from whole blood. Integration with centrifugal microfluidic technology allows us precise control of fluids while also reducing the expensive reagent consumption, the required analysis time and possible handling errors. We believe the presented result will not only improve the performance of the point-of-care-diagnostic devices but also potentially have great impact on global healthcare.

A Billion-Droplet AC Electrospray Digital PCR Platform for Large-Dynamic Range Nucleic Acid Quantification
Hsueh-Chia Chang, Bayer Professor of Chemical and Biomolecular Engineering, University of Notre Dame, Interim Chief Technology Officer, Aopia Biosciences, United States of America

An AC-electrospray technology is shown to be able to generate 1 billion femto-liter (micron diameter) aqueous drops in immiscible silicone oil in less than 20 minutes.  Unlike DC sprays, which tend to suffer from dielectric breakdown in liquid, and droplet generation technologies based on hydrodynamic shear, which can only generate pico-liter drops, we have shown that a properly tuned AC field can entrain low-mobility anions at a meniscus and the Coulombic repulsion among such entrained ions can deform the meniscus into unique 11 degree AC cones that are quite distinct from and much stable than DC cones (Phys Rev Lett, 101, 204501(2008); 109, 224301(2012); LabChip, 15, 1656(2015)).   The sharper AC cones can generate 1 million femto-liter drops per second.  The larger drop number and concentration (nM) allow us to get significantly better accuracy (without using Poisson statistics), dynamic range (8 decades) and reduction of inhibition and interfering effects in heterogeneous media.  We have fabricated an integrated AC electrospray chip with PCR and imaging modules and have favorably compared the performance of this new droplet digital PCR platform against qPCR. The new AC droplet digital PCR platform is, however, a low-cost turn-key technology that requires no or minimum sample pretreatment.

Cost-Effective Microfluidic Platform for Point-of-Care Diagnostics and Various Life-Science Applications
Alexander Govyadinov, Senior Technologist, HP Incorporated, United States of America

Recently, there is a lot of interest in microfluidic lab-on-a chip application for life science, forensic, point-of-care, molecular-diagnostic, other in-vitro-diagnostic, organs-on-a-chip, environmental and multiple others applications. Different scientific and commercial organizations explore a multitude of material sets and operational principles to forge microfluidic devices. Simultaneously, inkjet industry utilizes well established materials and principles of operation for complicated microfluidic systems developed for precision dispensing and manipulation of droplets with pico-liter accuracy on a massively parallel scale. The presentation describes our recent progress of low cost microfluidic platform development utilizing materials and processes developed for low-cost thermal inkjet business. The concept repurposes well established inkjet processes, microfluidic components and jetting elements for pumping, mixing, valving, fluid transport, sensing and other critical functions of complex integrated microfluidic systems.  This presentation describes operating principles of microfluidic elements, examples of their integration in functional devices and discusses inkjet technology potential for broad range of microfluidic applications.

The Art of 3D-Printing Biocompatible Microfluidics
Albert Folch, Professor of Bioengineering, University of Washington, United States of America

The vast majority of microfluidic systems are presently built by replica-molding and bonding in elastomers (such as poly(dimethyl siloxane) (PDMS)) or in thermoplastics (such as poly(methyl methacrylate) (PMMA) or poly-styrene (PS)). However, biologists and clinicians typically do not have access to microfluidic technology because they do not have the engineering expertise or equipment required to fabricate and/or operate microfluidic devices. Furthermore, the present commercialization path for microfluidic devices is usually restricted to high-volume applications in order to recover the large investment needed to develop the plastic molding processes. We are developing microfluidic devices through stereolithography, a form of 3D printing, in order to make microfluidic technology readily available via the web to biomedical scientists. Most available SL resins do not have all the favorable physicochemical properties of the above-named plastics (e.g., biocompatibility, transparency, elasticity, and gas permeability), so the performance of SL-printed devices is still inferior to that of equivalent PDMS devices. Inspired by the success of hydrogel PEG-DA biocompatibility, we have developed microfluidic devices by SL in resins that share all the advantageous attributes of PDMS and thermoplastics so that we can 3D-print designs with comparable performance and biocompatibility to those that are presently molded.

Microfluidic Printing: From Combinatorial Drug Screening to Artificial Cell Assaying
Tingrui Pan, Professor, Department of Biomedical Engineering, University of California-Davis, United States of America

Microfluidic impact printing has been recently introduced, benefiting from the nature of simple device architecture, low cost, non-contamination, scalable multiplexability and high throughput.  In this talk, we will review this novel microfluidic-based droplet generation platform, utilizing modular microfluidic cartridges and expandable combinatorial printing capacity controlled by plug-and-play multiplexed actuators.  Such a customizable microfluidic printing system allows for ultrafine control of the droplet volume from picoliters (~10pL) to nanoliters (~100nL), a 10,000 fold variation. The high flexibility of droplet manipulations can be simply achieved by controlling the magnitude of actuation (e.g., driving voltage) and the waveform shape of actuation pulses, in addition to nozzle size restrictions. Detailed printing characterizations on these parameters have been conducted consecutively. A multichannel impact printing system has been prototyped and demonstrated to provide the functions of single-droplet jetting and droplet multiplexing as well as concentration gradient generation. Moreover, several enabling chemical and biological assays have been implemented and validated on this highly automated and flexible printing platform.  In brief, the microfluidic impact printing system could be of potential value to establishing multiplexed droplet assays for high-throughput life science researchers.

Nanopore Sequencing for Real-Time Pathogen Identification
Kamlesh Patel, R&D Advanced System Engineering and Deployment Manager, Sandia National Laboratories, United States of America

As recent outbreaks have shown, effective global health response to emergent infectious disease requires a rapidly deployable, universal diagnostic capability. We will present our ongoing work to develop a fieldable device for universal bacterial pathogen characterization based on nanopore DNA sequencing. Our approach leverages synthetic biofunctionalized nanopore structures to sense each nucleotide. We aim to create a man-portable platform by combining nanopore sequencing with advance microfluidic-based sample preparation methods for an amplification-free, universal sample prep to accomplish multiplexed, broad-spectrum pathogen and gene identification.

Polymer-based Nanosensors using Flight-Time Identification of Mononucleotides 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 sequencing platform that can acquire sequencing information with high accuracy. 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 (~20 nm in width and depth; >100 µm in length) fabricated in a thermoplastic via nano-imprinting (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-less fashion by measuring current transients induced a single mononucleotide when it travels through a constriction with molecular dimensions (<10 nm in diameter) that are poised at the input/output ends of the flight tube. In this presentation, our efforts on building these polymer 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. Finally, information on the manipulation of single DNA molecules using nanofluidic circuits will be discussed that takes advantage of forming unique nano-scale features to shape electric fields for DNA manipulation and serves as the functional basis of the nanosensing platform.

Rapid and Ultra-sensitive Diagnostics Using Digital Detection
Weian Zhao, Associate Professor, Department of Biomedical Engineering, University of California-Irvine, United States of America

We will present our most recent droplet based digital detection platforms for rapid and sensitive detections, which could find potential applications at the point-of-care (POC).

Fractionation and Analysis of Nuclear versus Cytoplasmic Nucleic Acids from Single Cells
Juan Santiago, Charles Lee Powell Foundation Professor, Stanford University, United States of America

Single cell analyses (SCA) have become powerful tools in the study heterogeneous cell populations such as tumors and developing embryos.  However, fractionating and analyzing nuclear versus cytoplasmic fractions of nucleic acids remains a challenge as these fractions easily cross-contaminate.  We present a novel microfluidic system that can fractionate and deliver nucleic acid (NA) fractions from the nucleus (nNA) versus the cytoplasm (cNA) from single cells to independent downstream analyses.  Our technique leverages a selective electrical lysis which disrupts the cell’s (outer) cytoplasmic membrane, while leaving the nucleus relatively intact. We selectively extract, purify, and preconcentrate cNA using isotachophoresis (ITP).  The ITP-focused cNA and nNA-containing nucleus are separated by ITP and fractionated at a bifurcation downstream and then extracted for off chip analyses. We will present example applications of this fractionation including qPCR and next generation sequencing (NGS) analyses of cNA vs. nNA.  This will include preliminary NGS analyses of nuclear vs. cytoplasmic RNA fractions to analyze gene expression and splicing.  We hypothesize that the robust and precise nature of our electric field control is amenable to further automation to increase throughput while removing manuals steps.

Chip-Scale Microfluidic Physiological Circulation 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

There has been a recent surge in the development of microphysiological systems and organ-on-a-chip for drug screening and regenerative medicine.  Over the years, drug screening has mostly been carried out on 2D monolayers in well plates and the drugs are not delivered through blood vessels as in vivo treatments. 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 vascularized 3D tissues and to also pump and perfuse the tissue vascular network for on-chip microcirculation.  This in vitro model system can be used to screen cancer drugs by mimicking the delivery of the drugs through capillary blood vessels.  On the other hand, microfluidics play an important role in the recent advances in liquid biopsy and the ability to specifically isolate and capture rare cells such as circulating tumor cells. These two technologies may go hand-in-hand to connect in vitro screening to in vivo screening with great potential in the development of personalized medicine.

Biological Imaging Using Microfluidics and Electrochemistry
Charles Henry, Professor and Chair, Colorado State University, United States of America

Chemical gradients drive many processes in biology, ranging from nerve signal transduction to ovulation to cancer metastasis. At present, microscopy is the primary tool used to understand these gradients by imaging both the gradients and the resulting cell motion. Microscopy has provided many important breakthroughs in our understanding of fundamental biology, but is limited due to the need to incorporate fluorescent molecules into biological systems through either labeling or genetic manipulations. To better understand some processes, there is a need to develop tools that can measure chemical gradient formation in biological systems that do not require fluorescent modification of the targets, can be multiplexed to measure more than one molecule, and are compatible with a variety of biological sample types, including in vitro cell cultures and ex vivo tissue slices. In response to this need, we have developed a high-density electrode array containing 8,192 individual electrodes to image release of electrochemically active metabolites like nitric oxide and norepinephrine from live tissue slices. The electrode array has a resolution of 30 µm and covers and area of 2 mm by 2 mm, easily large enough to image release of neurotransmitters across an ex vivo tissue slice from a mouse model. In this talk, electrochemical characterization of this system will be discussed first using simple chemical models. Then use of the system in combination with microfluidics, for imaging spatiotemporal resolution of neurotransmitter release at the organ scale will be presented. Use of microfluidics to deliver stimulants that induce changes in release profile will be shown.

Human “Body-on-a-Chip” Devices as Tool to Improve Drug Development
Michael Shuler, Samuel B. Eckert Professor of Engineering, Cornell University, President Hesperos, Inc., United States of America

Alternatives or supplements to the use of animals in preclinical drug development that better mimic human response should reduce costs and increase the number of FDA approved drugs at the end of clinical trials. We have constructed micro-physiological (or “Body-on-a-Chip”) devices constructed from a combination of human tissue engineered constructs, micro-fabricated devices and physiologically based pharmacokinetic (PBPK) models. These human surrogates are constructed on a low cost, robust “pumpless” platform. In addition to measuring viability and metabolic responses, we can measure functional outputs such as electrical activity and force generation using integrated sensors (in collaboration with J. Hickman, University of Central Florida). We will focus our discussion on development of key organ modules and their integration with each other to form a model of the human body.