Micro & Nanotechnologies For Physical Phenotyping of Cells
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

My lab uses microtechnology to interface at the scale of biology to aid in scientific investigation, develop new approaches to diagnose and monitor disease, and engineer therapies. A part of our laboratory develops tools to assay and exploit physical properties of cells in diagnostics and drug screening. Physical properties of cells can provide integrative, rapid, and low-cost information about disease. My discussion will focus on instruments we have developed that quantify single-cell physical phenotypes such as deformability, size, and contractility. These new tools show promise to enable diagnostic and screening approaches that assay immune cell function at the point-of-care, tumor cell malignancy with higher confidence than molecular markers alone, and many other cell phenotypes associated with disease.

Re-envisioning Point-of-Care Pathogen Diagnostics for the Developed and Developing Worlds
Paul Yager, Professor, Department of Bioengineering, University of Washington, United States of America

Whether the purpose is 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 free testing from the centralized laboratory.  “Ubiquitous diagnostics”, aided by the revolution that brought us today’s distributed computing and communications, can bring the best diagnostic capabilities to physicians’ office laboratories and pharmacies in the developed world, or to places in the developing world where nothing is available now.  The practice of medicine itself can be improved if diagnostic tests could be carried out more rapidly and pervasively.  The Yager lab has been engaged in a 20-year pursuit of microfluidics-based tools and complete systems for pathogen identification in human samples, most recently in an inexpensive instrument-free single-use disposable format that could be used by consumers in their homes and by healthcare workers in low-resource settings in the developing world.  The central effort today is to utilize capillary action in porous materials to eliminate the need for pumps and other equipment to control fluid flow.  By coupling the chemical testing with cell phones, critical health data can be analyzed rapidly and anywhere, and the best healthcare decisions can be made for patients, for regional healthcare systems, and for global health.  There have been 3 analytical approaches pursued in our group:  Identification and quantification of 1) antibodies specific to pathogens, 2) proteins of the pathogens, and 3) nucleic acid sequences derived from the pathogens.  We report on recent results on particularly simple paper-based systems supported by NIH (detection of influenza proteins; improvement of specificity of Zika virus serology), DARPA (detection of DNA and RNA from bacterial and viral pathogens) and DTRA (detection of proteins from the Ebola virus).  All projects involve close collaboration with partners inside and outside the university environment, and are aimed at producing clinically useful tools for point-of-care medicine.

Paper-based Nanobiosensors: Diagnostics Going Simple
Arben Merkoçi, ICREA Professor and Director of the Nanobioelectronics & Biosensors Group, Institut Català de Nanociencia i Nanotecnologia (ICN2), Barcelona Institute of Science and Technology (BIST), Spain

Biosensors field is progressing rapidly and the demand for cost efficient platforms is the key factor for their success. Physical, chemical and mechanical properties of cellulose in both micro and nanofiber-based networks combined with their abundance in nature or easy to prepare and control procedures are making these materials of great interest while looking for cost-efficient and green alternatives for device production technologies. Both paper and nanopaper-based biosensors are emerging as a new class of devices with the objective to fulfill the “World Health Organization” requisites to be ASSURED: affordable, sensitive, specific, user-friendly, rapid and robust, equipment free and deliverable to end-users. How to design simple paper-based biosensor architectures? How to tune their analytical performance upon demand? How one can ‘marriage’ nanomaterials such as metallic nanoparticles, quantum dots and even graphene with paper and what is the benefit?  How we can make these devices more robust, sensitive and with multiplexing capabilities? Can we bring these low cost and efficient devices to places with low resources, extreme conditions or even at our homes? Which are the perspectives to link these simple platforms and detection technologies with mobile phone communication? I will try to give responses to these questions through various interesting applications related to protein, DNA and even contaminants detection all of extreme importance for diagnostics, environment control, safety and security.

Microfluidic Devices – Key Technologies to Enable Real-Time Patient Monitoring and Treatment
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

Clinical practice is beginning to wake upto the potential of real-time molecular information from venerable tissue as a means to understand the progression in the tissue of injury or disease. Such patterns of molecular changes, particularly when combined with paternal of physical of electrical signatures, offer the exciting possibility of allowing clinicians to guide therapy on an individualized basis in real time. In this presentation I will describe the development of 3D printed microfluidic devices connected to wireless electronics for transplant organ and patient monitoring. Tissue sampling is via integrated microdialysis probes. Concentrations of important biomarkers are measured using microscale amperometric biosensors (energy metabolites, and excitatory neurotransmitters) and ion-selective electrodes (ISE) for tissue ionic balance. Detailed patterns of ionic responses can be revealed using a high density ISE array within the flow stream. High time resolutions can be achieved using a novel droplet based microfluidic system. The presentation with describe the design and optimization challenges and include clinical examples from our recent work.

Microfluidics for Bottom-Up Tissue Engineering
Shoji Takeuchi, Professor, Center For International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science, The University of Tokyo, Japan

In this presentation, I will talk about several Microfluidic-based approaches for the rapid construction of 3D-cellular construct. Large-scale 3D tissue architectures that mimic microscopic tissue structures in vivo are very important for not only in tissue engineering but also drug development without animal experiments. We demonstrated a construction method of 3D tissue structures by using cell beads and cell fibers. To prepare the cellular beads, we used an axisymmetric flow focusing device (AFFD) that allows us to encapsulate cells within monodisperse collagen beads. By molding these cell beads into a 3D chamber and incubating them, we successfully obtained complicated and milli-sized 3D cellular constructs. As the cell fibers, a cell-encapsulating core-shell hydrogel fiber was produced in a double coaxial laminar flow microfluidic device. When with myocytes, endothelial, and nerve cells, they showed the contractile motion of the myocyte cell fiber, the tube formation of the endothelial cell fibers and the synaptic connections of the nerve cell fiber, respectively. By reeling, weaving and folding the fibers using microfluidic handling, higher-order assembly of fiber-shaped 3D cellular constructs can be performed. Moreover, the fiber encapsulating beta-cells is used for the implantation of diabetic mice, and succeeded in normalizing the blood glucose level.

Mixed-Scale Fluidic System: Searching for Drug-induced DNA Damage in Circulating Tumor Cells
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

Improved therapies that yield more cures and better overall survival for cancer patients are needed. For example, women with breast cancer have a 5-year survival rate of 22% (Stage IV) and 72% (Stage III). Doxorubicin, cisplatin, paclitaxel, and tamoxifen are examples of drugs used for treating breast cancer with selection of therapy typically based on the classification and staging of the patient’s cancer. While treatment regimens assigned to some patients may be optimal using the current classification model, others within certain breast cancer sub-types fail therapy. New assays must be developed to determine how a patient’s physiology and genetic makeup affects drug efficacy. In this presentation, a series of chips are used for the isolation and processing of circulating tumor cells (CTCs). The chips quantify response to therapy using three pieces of information secured from the CTCs; (1) CTC number; (2) CTC viability; and (3) the frequency of DNA damage (abasic (AP) sites) in genomic DNA (gDNA) harvested from the CTCs. Microscale chips are used for CTC selection, CTC enumeration and viability determinations. The chip to read AP sites is a nanosensor chip made via nano-imprinting in plastics and contains a nanochannel with dimensions less than the persistence length of double-stranded DNA (~50 nm). Labeling AP sites with fluorescent dyes and stretching the gDNA in the nanochannel to near its full contour length allows for the direct readout of the AP sites, even from a single CTC. This information is used to determine how a patient is responding to therapy.

PCR-Free MicroRNA Quantification Based on Ion-Selective Nanoporous Membranes and Nanopores
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 an integrated biochip platform that can identify and quantify low copy numbers of microRNA biomarkers in a heterogeneous physiological sample like blood, saliva or urine. This quantification assay is done without PCR amplification, reporter labeling,  extensive off-chip pretreatment and expensive optical sensors.  Consequently, it does not introduce PCR/ligation bias and limits analyte loss during pretreatment.  The main components of the integrated biochips are nanoporous membranes and solid-state nanopores with pore radii smaller than the nm-scale Debye length. We use the ion concentration and charge polarization features of the ion-selective membranes to control the on-chip ionic strength, actuate pH by splitting water, lyse exosomes and isolate/concentrate the target molecules. The final nanopore sensor or sensor array utilizes surface modification and pore geometry to preferentially delay the translocation time of the target microRNAs to achieve single-molecule identification and quantification. The integrated chip achieves a translocation frequency (throughput) that is at least one hundred times higher than any literature or commercial nanopore technology.

Nanomotor-Microchip Diagnostics: Moving the Receptor in Microchannel Networks
Joseph Wang, Chair of Nanoengineering, SAIC Endowed Professor, Director at Center of Wearable Sensors, University of California-San Diego, United States of America

This presentation will describe new motion-based microchip assays based on autonomously moving receptor-functionalized nanomotors. The new motor-based sensing approach relies on new capabilities of modern nano/microscale motors. Particular attention will be given to catalytic nanowire and microtube motors propelled by the electrocatalytic decomposition of a chemical fuel,  The increased cargo-towing force of new man-made nanomotors, along with their precise motion control within microchannel networks, versatility and facile functionalization, can be combined for developing advanced microchip systems based on active transport. The motion-based biosensing strategy relies on the continuous movement receptor-modified microengines through complex samples in connection to diverse ‘on-the-fly’ biomolecular interactions of nucleic acids, proteins, bacteria or cancer cells. A variety of receptors, attached to self-propelled nanoscale motors, can thus move around the sample and, along with the generated microbubbles, lead to greatly enhanced fluid transport and accelerated recognition process. Selective capture and transport of target DNA and cancer cells from raw complex body fluids will be demonstrated. Key factors governing such motion-based sensing will be covered. The resulting assays add new and rich dimensions of analytical information and offer remarkable sensitivity, coupled with simplicity, speed and low costs. We will discuss the challenges of implementing molecular recognition into the nanomotor movement and for generating well-defined distance signals. New microengines with a ‘built-in’ recognition capability, based on boronic-acid or molecularly-imprinted outer layers, will also be discussed. The latter obviated the need for the receptor immobilization. The greatly improved capabilities of chemically-powered artificial nanomotors could pave the way to exciting and important bioanalytical applications and to sophisticated nanoscale and microchip devices performing complex tasks.

Correlated Chemical Imaging and Spatiotemporal Organization in Microbial Communities
Paul Bohn, Arthur J. Schmitt Professor of Chemical and Biomolecular Engineering and Professor of Chemistry and Biochemistry, University of Notre Dame, United States of America

Biofilms, such as those formed by the opportunistic human pathogen Pseudomonas aeruginosa are complex, matrix-enclosed, surface-associated communities of cells that exhibit properties - such as enhanced resistance to antibiotics - distinct from their free-floating counterparts. P. aeruginosa biofilms are associated with persistent and chronic infections in diseases such as cystic fibrosis and HIV-AIDS and thus are primary targets for the lab-on-a-chip community. P. aeruginosa cells organize themselves by synthesizing and secreting signaling molecules, implicated in quorum sensing (QS), and in regulating biofilm formation and virulence. Correlated chemical imaging using powerful molecular imaging platforms, such as confocal Raman microscopy and SIMS-based mass spectrometric imaging, in conjunction with multivariate statistical tools, are being applied to study the spatial and temporal distributions of signaling molecules, secondary metabolites and virulence factors in biofilm communities of P. aeruginosa. These studies reveal that laboratory strains of Pseudomonas differ significantly both from genetic mutants and from clinical isolate strains in the mechanisms used for chemical communication as well as the organization of the resulting biofilms in monoculture.  They also reveal significant modes of action in co-cultures involving different strains and different bacterial species.

Microfluidic and 3D Printing Devices for Cell Patterning and Tumor Analysis
Xueji Zhang, Professor & Director, University of Science and Technology Beijing, China

Traditional methods for living cell analysis have been limited by the incapability of gaining information from dynamic imaging and molecular level information such as gene expression simultaneously. Herein we report an easy but versatile method for patterning different cells on a single substrate by using a microfluidic approach “Ip-Do Assay”, which allows not only controlling of cells niches but also retrieval of specific treated cells to quantitatively analysis their gene expression. Recently, we also developed a 3D printed “LEGO”-like device to implement tumor cell migration and metastasis assay without any micro-fabrication process due to the easy configuration of 3D printing. This technology allows quantitative analysis on not only tumor cell migration at single cell level but also for tumor-somatocyte interactions.