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"""Virtual vials"" for enhanced biomolecular analysis"

Final Report Summary - VIRTUALVIALS ("Virtual vials" for enhanced biomolecular analysis)

Virtual Vials is an EID project between Technion and IBM–Zurich, aimed at developing novel tools for biomolecular analysis and training the next generation of scientists and engineers who likely will translate the technologies and discoveries into practical use for the benefit of society and the EU hi-tech sector. In vitro diagnostics is experiencing critical shifts in paradigms with a growing market which encompasses clinical laboratory tests, point-of-care devices, and basic research aimed at understanding the origin of diseases and developing improved testing and treatments. The development of novel tools, which enable increased analysis speeds, higher sensitivity, higher throughput, miniaturization, and portability are essential for enabling discoveries and providing better care for patients. Our project includes three distinct yet synergetic projects for advancing biomolecular analysis. Each project is intended as a PhD project that integrates specific skills and know-how from Technion and IBM. The uniting theme of the projects is the use of microfluidics and isotachophoresis (ITP) to enable precise transport, focusing, and control of samples within micron-sized channels.

During the project, we have made major advances in each of the above three projects.

Isotachophoresis-based surface immunoassay: Surface immunoassays (e.g. ELISA, lateral flow) are the gold standard in clinical and point-of-care diagnostics. In contrast to detection of nucleic acid sequences where amplification methods exist, detection of proteins must rely solely on the initial concentration of targets in the sample. Therefore, at low concentrations, immunoassays are limited by the slow binding kinetics between targets and antibodies. We demonstrated the use of isotachophoretic focusing for acceleration of antibody-protein binding kinetics, enabling a significant reduction in limit of detection (LoD) compared to standard immunoassays. Our assay is based on using ITP to deliver the focused sample to a surface functionalized with capture antibodies, where the locally high target concentration boosts the reaction. We distinguish between two modes of operation: continuous ITP, where the sample reacts with the surface while electro-migrating downstream, and stop-and-diffuse ITP, where the electric field is temporarily stopped, enabling longer reaction times.

Delivery of minimally dispersed liquid interfaces for sequential surface chemistry using a microfluidic probe: Delivery of multiple reagents in sequence is central in a large number of surface biochemical assays. For example, protein interactions and function analysis, cell stimulation response, signaling and chemical communication studies, and nucleic acid hybridization- and immuno-assays all require delivery of multiple reagents to a reaction site in a fixed order. We developed a novel method which allows contact-free sequential delivery of reagents to a reaction surface, with a characteristic switching time of 560 ms over a transport distance of 60 cm. The method is implemented on a vertical microfluidic probe (vMFP) — a non-contact scanning device that hydrodynamically confines liquids to a nanoliter-scale volume between an injection and an aspiration channel at its apex. This confined flow can be brought into contact with any surface, including cell tissues, to drive a reaction. Owing to the contact-free operation of the MFP, multiple reaction sites on the surface can be addressed automatically.

Focusing 10 uL into 500 pL - on-chip processing of large volumes using isotachophoresis: A significant limitation of many microfluidics-based diagnostics assays remains their inability to process sufficiently large sample volumes. This is particularly important for cases where the biological target exists at such low concentrations that its presence in the microchannel becomes probabilistic. We developed a novel microfluidic chip capable of focusing a target analyte from sample volumes on the order of 10 uL into 500 pL volumes using isotachophoresis. We demonstrated the potential of this approach for highly sensitive detection of both biomolecular targets (DNA, down to 100 fM) and of discrete targets (whole bacteria, down to 1000 cfu/ml).

Microscale flow control using non-uniform electro-osmotic flow: We developed a novel method that allows establishing desired flow patterns in a Hele-Shaw cell, solely by controlling the surface chemistry, without the use of physical walls. Using weak electrolytes, we locally pattern the chamber's ceiling and/or floor, thus defining a spatial distribution of surface charge. This translates to a non-uniform electric double layer which when subjected to an external electric field applied along the chamber, gives rise to non-uniform electroosmotic flow (EOF). We developed the theory that allows prediction and design of such flows fields and demonstrated them experimentally, opening the door to configurable microfluidic devices.

Electrokinetic scanning probe for biomolecular analysis: The ability to electrically control the delivery and extraction of ionic species and fluids to and from localized regions on standard biological substrates in the “open space” immersed in liquids opens the door to a range of new applications in deposition and extraction of biomolecules on surfaces. We developed a new concept for a scanning probe, in which transport of fluid and molecules is driven solely by an electric field. We term this device an electrokinetic scanning probe (ESP). The electrokinetic probe can be operated in one of two modes, or their combination: (1) Electroosmotic flow (EOF) mode, where EOF serves as the driving mechanism for the fluid; By adjusting the electric field in each of the channels, the relative flow rates can be controlled, enabling conditions similar to those obtained in pressure driven microfluidic probes, where confinement of the injected reagents is achieved at the tip. (2) Electrophoretic mode, where EOF is suppressed and no fluid motion occurs; analytes are electrophoretically transported from the injection to the aspiration channel, passing through the open fluid.

Potential outcome of the virtual vials project and economical value:
• In vitro diagnostics is a rapidly growing field and market which encompasses clinical laboratory tests, point-of-care devices, as well as basic research aimed at understanding the origin of diseases and developing improved testing and treatments.
• The 2015 global market for in vitro diagnostics was estimated at $56 billion, and is projected to grow to $75 billion by 2020.
• The development of novel tools, which enable increased analysis speeds, higher sensitivity, higher throughput, miniaturization, and portability are essential for enabling discoveries and providing better care for patients. Within this project, we aim to develop new methods and tools that will enable a new class of highly sensitive point-of-care devices.

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