Point-of-Care Diagnostics & Biosensors Europe 2018 Agenda

Conference Chairman Welcome and Opening Remarks
Richard Chasen Spero, CEO, Redbud Labs, United States of America

Nanoplasmonic Biosensors: From Innovative Materials to Multimode Sensing with Integrated Microdevices
Amy Shen, Professor and Provost, Okinawa Institute of Science and Technology Graduate University, Japan

Gold nanostructures are a highly attractive class of materials with unique electrochemical and optical sensing properties. Recent developments have greatly improved the sensitivity of optical sensors based on metal nanostructured arrays. We introduce the localized surface plasmon resonance (LSPR) sensors and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events. We then describe recent progress in three areas representing the most significant challenges: integration of LSPR with complementary electrochemical techniques, long term live-cell biosensing and practical development of sensors and instrumentation for routine use and high-throughput detection. As an example we will demonstrate a novel refractive index and charge sensitive device integrated with nanoplasmonic islands to develop nano-metal-insulator-semiconductor (nMIS) junctions. The developed sensor facilitates simultaneous detection of charge and mass changes on the nanoislands due to biomolecule binding. A brief insight on microcontact printing to functionalize proteins on nanoplasmonic sensors will also be discussed. The developed nanosensors can readily be adopted for multiplexed and high throughput label-free immunoassay systems, further driving innovations in biomedical and healthcare research.

Towards Wearable Real-Time Clinical Monitoring Using Microfluidic Devices
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

Modern acute critical care medicine is increasingly seeking to protect vulnerable tissue from damage by monitoring the patterns of physical, electrical and chemical changes taking place in tissue – so called multimodal monitoring. Such patterns of molecular changes offer the exciting possibility of allowing clinicians to detect changes in patient condition and to guide therapy on an individualized basis in real time. 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. In this presentation, I will describe the combination of miniature electrochemical sensors and biosensors with 3D printed microfluidic devices for transplant organ and patient monitoring. Concentrations of key biomarker molecules can then be determined continuously using either optically or electrochemically, using amperometric, potentiometic and array 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.

Silicon Nanotechnology Meets Biology (Smaller and Wetter is Better)
Gregory Timp, Keough-Hesburgh Professor of Electrical Engineering & Systems Biology, The University of Notre Dame, United States of America

According to Moore’s law, the scaling of silicon integrated circuits is supposed to reach the 5 nm-node sometime after 2020, although the schedule is still problematic due to the astronomical cost and atomically precise line-rules. On the other hand, biology has been performing cost-effectively using proteins the size of 5 nm (and smaller) that fold with atomic precision for 4.28 billion years now—it is a robust and proven technology, albeit wet. In this talk, it is argued that there is still “plenty of room at the bottom” for improving performance if silicon nanotechnology is adapted to biology. With silicon nanotechnology it is now within our grasp to create an interface to biology on a nanometer-scale. Three examples of such interfaces are proffered. The first is a liquid flow cell that works like an envelope made from 30 nm-thick silicon nitride membranes, which can hold and sustain living cells in medium and yet fits inside a Scanning Transmission Electron Microscope (STEM). In a STEM, the liquid cell can be used to visualize and track live cell physiology like a phage infecting a bacterium with nucleic acids at 5 nm resolution. The second is a nanometer-diameter pore sputtered through a silicon nitride membrane 10-nm-thick that can be used to transfect cells precisely with nucleic acids to affect gene expression in them and, under different bias conditions, detect protein secretions from single cells with single molecule sensitivity. The secretions inform on the cell phenotype and offer a molecular diagnosis of disease. Finally, the third interface is a sub-nanometer-diameter pore, which is about the size of an amino acid residue, in either silicon dioxide or silicon nitride membranes ranging from 6 to 10 nm-thick.  Sub-nanopores like this have been used to read the primary structure of a protein, i.e. the amino acid sequence, with low fidelity, but with single molecule sensitivity, vastly outstripping the sensitivity of conventional methods for sequencing such as mass spectrometry. Taken altogether, the prospects are dazzling for a new type of integrated circuit that incorporates biology with state-of-the-art silicon electronics.

Extracellular Vesicles (EVs) and Cell Free DNA (cfDNA) as Blood-based Biomarkers: Plastic-based Microfluidics for their Enrichment and Analysis
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

While there are a plethora of different blood-based markers, EVs are generating significant interests due to their relatively high abundance (~10¹³ particles per mL of blood) and the information they carry. EVs contain a diverse array of nucleic acids, such as mRNA, lncRNA, and miRNA that can be used for disease management. In addition to EVs, cfDNA also are biomarkers that can be used to help manage different disease states using the mutations they possess that can have high diagnostic value. In spite of the relatively high abundance of cfDNA in diseased patients (~160 ng/mL), the extraction and enrichment of cfDNA has been inefficient, even by commercial kits, due to the low abundance of the tumor bearing DNA fragments (<0.01%) and the short nature of these fragments, especially cancer-related cfDNA (as small as 50 bp). In this presentation, we will discuss the design, fabrication and analytical figures-of-merit of a microfluidic device that can serve the dual purpose for the affinity-based selection of EVs and the solid phase extraction of cfDNA directly from plasma using the same device. The microfluidic is made from a plastic that can be injection molded to produce high quality devices at low cost. For EVs, the device is made cyclic olefin copolymer (COC) is UV/O3 activated to allow for the efficient immobilization of affinity agents to the surface of the device. In the case of cfDNA, the device is made from COC as well, but is only UV/O3 activated (i.e., no affinity agents used). Information will be provided as to the ability to molecularly profile the cargo contained within the affinity-selected EVs, in particular mRNA expression profiling. We will also discuss the use of this microfluidic to isolate with high recovery cfDNA from plasma samples with size selection capabilities. The isolated cfDNA could be queried for mutations using an allele-specific ligation detection reaction at a mutant to wild-type ratio <0.1%.

Microphysiological Models Relying on Emergence of Multi-Cellular Engineered Living Systems
Roger Kamm, Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering, Massachusetts Institute of Technology (MIT), United States of America

Recent work from many labs has demonstrated the unique capability of cells placed in 3D culture to self-organize into functional units and organ-like systems.  In some cases, pluripotent cells can be induced to differentiate down independent pathways, leading to an organoid.  In others, interacting units can be generated, often from iPS cells, and ‘engineered’ to interact in a way that recapitulates certain aspects of in vivo function or disease.  Such models have tremendous potential both to gain new insight into disease processes and for moderate throughput drug screening.  In this talk I will describe several models developed in our lab including a neuromuscular junction, blood-brain barrier, and vascularized skeletal muscle, addressing some of the design principles they have in common, the future potential, and barriers to progress.

Microscale Flow Patterning
Moran Bercovici, Associate Professor, Faculty of Mechanical Engineering; Head, Technion Microfluidic Technologies Laboratory, Technion, Israel Institute of Technology, Israel

The ability to manipulate fluids at the microscale is a key element of any lab-on-a-chip platform, enabling core functionalities such as liquid mixing, splitting and transport of molecules and particles. Lab-on-a-chip devices are commonly divided in two main families: continuous phase devices, and discrete phase (droplets) devices. While a large number of mechanisms are available for precise control of droplets on a large scale, microscale control of continuous phases remains a substantial challenge. In a traditional continuous-flow microfluidic device, fluids are pumped actively (e.g. by pressure gradients, electro-osmotic flow) or passively (e.g. capillary driven) through a fixed microfluidic network, making the device geometry and functionality intimately dependent on one another (e.g. DLD, inertial mixer, H-separator, etc.). The advent of on-chip microfluidic valves brought more flexibility in routing fluids through microfluidic networks, adding a dynamic dimension to the static geometrical network. However, the number of degrees of freedom of valve-based systems is restricted by their dependence on bulky pneumatic lines (regulators, pressure systems, controllers), which are difficult to scale down in size and cost. In this talk I will present our ongoing work leveraging non-uniform EOF and thermocapillary flows to control flow patterns in microfluidic chambers.  By setting the spatial distribution of surface potential or a spatial temperature distribution, we demonstrate the ability to dictate desired flow patterns without the use of physical walls. We believe that such flow control concepts will help break the existing link between geometry and functionality, bringing new capabilities to on-chip analytical methods.

Integrating Aptamer Technology with Paper-Based Point-of-Care Devices for Biomedical Monitoring
John Brennan, Professor and Director, Biointerfaces Institute, McMaster University, Canada

The talk will focus on the development of printed point-of-care paper sensors with integrated aptamer-based recognition, amplification and signaling technologies for detection of a range of clinical analytes.  Examples will be provided to demonstrate multi-step reactions on paper for ultra-sensitive detection of infectious organisms and cancer biomarkers.

Point-of-Care Diagnostics 2.0
Emmanuel Delamarche, Manager Precision Diagnostics, IBM Research - Zürich, Switzerland

Diagnostics are ubiquitous in healthcare because they support prevention, diagnosis and treatment of diseases. Specifically, point-of-care diagnostics are particularly attractive for identifying diseases near patients, quickly, and in many settings and scenarios. One of our contribution to the field of microfluidics is the development of capillary-driven microfluidic chips for highly miniaturized immunoassays. In this presentation, I will review how to program capillary flow and encode specific functions to form microfluidic elements that can easily be assembled into self-powered devices for immunoassays, reaching unprecedented levels of precision for manipulating samples and reagents. This technology can also be augmented using peripherals and smartphones for flow control and monitoring with sub-nanoliter precision. Is the next generation of point-of-care devices finally coming?

Nanobiosensors Design and Applications in Diagnostics
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

There is a high demand to develop innovative and cost effective devices with interest for health care beside environment diagnostics, safety and security applications. The development of such devices is strongly related to new materials and technologies being nanomaterials and nanotechnology of special role. We study how new nanomaterials such as nanoparticles, graphene nano/micromotors can be integrated in simple sensors thanks to their advantageous properties. Beside plastic platforms 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 fulfil 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 couple 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 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.

Novel Isolate of Extracellular Nanovesicles
Jong Wook Hong, Professor of Bionano Technology and Bionano Engineering, Hanyang University, Korea South

Extracellular vesicles (EVs) are the cell-secreted nano- and micro-sized particles consisted of lipid bilayer containing nucleic acids and proteins for diagnosis and therapeutic applications. The inherent complexity of EVs is a source of heterogeneity in various potential applications of the biological nanovesicles including analysis. To diminish heterogeneity, EV should be isolated and separated according to their sizes and cargos. However, current technologies do not meet the requirements. Here we introduce new way that provides noninvasive and precise separation of EVs based on their sizes without any recognizable damages. We believe the system and methodology would open new windows in precision medicine and personalized medicine.

Single Molecule Screening
Joshua Edel, Professor, Imperial College London, United Kingdom

Analytical Sensors play a crucial role in today’s highly demanding exploration and development of new detection strategies. Whether it be medicine, biochemistry, bioengineering, or analytical chemistry the goals are essentially the same: 1) improve sensitivity, 2) maximize throughput, 3) and reduce the instrumental footprint. In order to address these key challenges, the analytical community has borrowed technologies and design philosophies which has been used by the semiconductor industry over the past 20 years. By doing so, key technological advances have been made which include the miniaturization of sensors and signal processing components which allows for the efficient detection of nanoscale object. One can imagine that by decreasing the dimensions of a sensor to a scale similar to that of a nanoscale object, the ultimate in sensitivity can potentially be achieved - the detection of single molecules.

High Content Single Cell Analysis Using the Programmable Bio-Nano-Chip System: New Tools for Cancer Diagnosis
John McDevitt, Chair, Department Biomaterials, New York University College of Dentistry Bioengineering Institute, United States of America

Over the past few decades, use of biomarkers has become increasingly intrinsic to practice of medicine and clinical decision-making. Diagnosis and management of oral cancer is a promising area whereby biomarker driven testing has potential to provide significant impact on patient care. Oral cancer is sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates with little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of oral cancer. This talk will feature details related to a new ‘cytology-on-a-chip’ platform capable of high-content single-cell measurements. This methodology permits concurrent analysis of molecular biomarker expression and cellular/nuclear morphology using over 200 fluorescence intensity and shape parameters for each region of interest extracted from multi-spectral fluorescence images. Molecular biomarkers: EGFR, avß6, CD147, ß-catenin, MCM2, and Ki67 were selected based on their capacity, through prior immunohistochemistry studies, to distinguish stages of disease progression towards oral cancer. Measurement time to complete this chip-based image analysis is approximately 20 minutes vs. about 1-3 days for gold standard pathology exam. This new clinical decision tool has been developed and validated in context of major clinical study involving 714 prospectively recruited patients. These efforts have led to collection of data across 6 diagnostic categories and assembly of one of largest well-qualified cytology database (confirmed by tissue biopsy) ever collected for prospectively recruited potentially malignant oral lesions. The application of statistical machine learning algorithms exploiting this large database has led to development of robust classification models with validated and stable parameters. High sensitivity and high specificity adjunctive diagnostic aids have been developed through these efforts.

A New Platform for Point-of-Care Testing of Cardiac Biomarkers Meeting Current Guidelines
Eloisa Lopez-Calle, Head Assay Formats, Roche Diagnostics GmbH, Germany

Cardiovascular diseases, such as acute myocardial infarction (AMI) and heart failure (HF), are the leading cause of death globally. A rapid and accurate diagnosis of these diseases is crucial for the immediate initiation of the treatment for the patients; and biomarkers, such as troponin and NT-proBNP, received the highest clinical guideline recommendation for that use. The measurement of these biomarkers with point-of-care (POC) devices has the unique benefits to (i) reduce the turn-around-time compared to laboratory testing, by avoiding the time-consuming process of sample transportation and pre-analytics (plasma/serum generation), (ii) to use it in the pre-hospital, ambulance settings, and (iii) to rule-out AMI/HF faster, thus allowing an improved flow of patients through the emergency department. Today, however, only few of the commercially available POC-systems show a guideline-compliant analytical performance for troponin testing, which is a recommended imprecision (coefficient of variation, CV) of <10% at the clinical cutoff, given as the 99th percentile upper reference limit of a healthy population.  We have developed an easy-to-use and portable prototype platform for the POC-testing of cardiac troponin T (cTnT) and NT-proBNP using a 30 µL whole blood sample with a time-to-result of 12 minutes or less. The immunoassay is run in a ready-to-use disposable cartridge with fully integrated reagents. After sample application of whole blood, plasma is generated by centrifugation, followed by incubation with immuno-reagents and washing. Finally, the fluorescence is measured, followed by calculating the biomarker amount. This presentation shows features of the new, CLIA-waivable platform matching with POC settings as well as the proof-of-concept for the guideline-compliant determination of cTnT and NT-proBNP with lab-like performance (cTnT: 3.8% as CV at the cutoff (14 ng/L), 6.8 ng/L as functional sensitivity (CV<10%), 8300 ng/L as upper-end measuring range; NT-proBNP:  4.1% as CV at 125 pg/mL-cutoff, 23 pg/mL as functional sensitivity, upper-end measuring range: 17000 pg/mL; excellent correlation of both assays with the commercial Elecsys® tests, hs-TnT 5th Gen. and proBNP II).