Innovations in Microfluidics & SCA 2022 Agenda

Precision Microfluidics of Large Volumes
Mehmet Toner, Helen Andrus Benedict Professor of Biomedical Engineering, Massachusetts General Hospital (MGH), Harvard Medical School, and Harvard-MIT Division of Health Sciences and Technology, United States of America

Microfluidics gained prominence with the application of microelectromechanical systems (MEMS) to biology in an attempt to benefit from the miniaturization of devices for handling of minute samples of fluids under precisely controlled conditions. Microfluidics exploits the differences between micro- and macro-scale flows, for example, the absence of turbulence, electro-osmotic flow, surface and interfacial effects, capillary forces in order to develop scaled-down biochemical analytical processes. The field also takes advantage of MEMS and silicon micromachining by integrating micro-sensors, micro-valves, and micro-pumps as well as physical, electrical, and optical detection schemes into microfluidics to develop the so-called “micro-total analysis systems” or “lab-on-a-chip” devices. However, the ability to process ‘real world-sized’ volumes efficiently has been a major challenge since the beginning of the field of microfluidics. This begs the question whether it is possible to take advantage of microfluidic precision without the limitation on throughput required for large-volume processing? The challenge is further compounded by the fact that physiological fluids are non-Newtonian, heterogeneous, and contain viscoelastic living cells that continuously responds to the smallest changes in their microenvironment. Our efforts towards moving the field of microfluidics to process large-volumes of fluids was counterintuitive and not anticipated by the conventional wisdom at the inception of the field. We metaphorically called this “hooking garden hose to microfluidic chips.”  We are motivated by a broad range of applications enabled by precise manipulation of extremely large-volumes of complex fluids, especially those containing living cells or bioparticles. This presentation will provide a summary of our efforts in bringing microfluidics to large volumes and complex fluids as well as various applications such as the isolation of extremely rare circulating tumor cells (CTCs) from whole blood.  The use of high-throughput microfluidics to process large-volumes of complex fluids (e.g., whole blood, bone marrow, bronchoalveolar fluid) has found broad interest in both academia and industry due to its broad range of utility in medical applications.

Plastic-based Nanofluidic Devices for Single Molecule DNA/RNA 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, amplification-free 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 nanochannels that read the identity of individual mononucleotides from their molecular-dependent electrophoretic mobilities through a 2-dimensional (2D) nanochannel (~50 nm in width and depth; >10 µm in length) fabricated in a thermoplastic via nanoimprint lithography (NIL) or injection compression molding. The single 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 cleaved mononucleotides into the 2D nano-electrophoresis channel. The identity of each mononucleotide is deduced from its molecular-dependent electrophoretic mobility through the 2D nanochannel. The mobility 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 (<10 nm in effective diameter) poised at the input/output ends of the electrophoresis channel.

In this presentation, our results in using nanoscale electrophoresis to deduce the identity of both the deoxynucleotides and ribonucleotides will be discussed, especially material and scaling effects on the performance of nano-electrophoresis. Also, different surface modification strategies of thermoplastics will be presented that alter the electroosmostic flow and its effects on separation performance. I will also discuss the surface immobilization of exonucleases onto solid-plastic supports using UV/O3 activation with EDC/NHS coupling chemistry. In particular, the effects of surface immobilization on enzyme kinetic rates, processivity, and stability will be discussed. Finally, the fabrication and operation of in-plane nanopore sensors to detect single molecules will be discussed.

mechano-NPS: An Electronic Method to Mechanically Phenotype Cells
Lydia Sohn, Almy C. Maynard and Agnes Offield Maynard Chair in Mechanical Engineering, University of California-Berkeley, United States of America

We have developed an efficient, label-free method of screening cells for their phenotypic profile, which we call Node-Pore Sensing (NPS). NPS involves measuring the modulated current pulse caused by a cell transiting a microfluidic channel that has been segmented by a series of inserted nodes. Previously, we showed that when segments between the nodes are functionalized with different antibodies corresponding to distinct cell-surface antigens, immunophenotyping can be achieved. In this talk, I will show how we have significantly advanced NPS by simply inserting between two nodes a “contraction” channel through which cells can squeeze. “Mechano-NPS”, as we now call our method, can simultaneously measure a cell’s size, stiffness, and ability to recover from deformation. We have used mechano-NPS to assess the mechanical properties of acute promyelocytic leukemia (APL) cells. APL is an acute myeloid leukemia subtype for which all-trans retinoic acid (ATRA) is an essential therapy. 20% of APL patients are resistant to ATRA, which must be administered during the acute phase of the disease to prevent death. We show that ATRA resistant APL cells are less mechanically pliable than ATRA-responsive cells. Thus, a potential biomarker for APL resistance may ultimately be mechanical stiffness.

On-Site Diagnostics in Resource-Limited Settings
Nicole Pamme, Professor in Analytical Chemistry, Stockholm University, Sweden

Our research centers on the study of microfluidic lab-on-a-chip devices applied to environmental analysis, biomedical research and the synthesis of smart materials. Microfluidic devices offer the possibility for in-the-field and point-of-care analysis provided the devices are portable, require only minimal external instrumentation and little power and are robust. In our group, we are investigating a simple to operate workflows for pathogen isolation from clinical and environmental matrices in collaboration with researchers in South Africa and Kenya. This has included projects on the analysis of pathogens in waste water, pathogens in maternal health monitoring and analysis of SARS-CoV-2 RNA.

Analyzing and Sorting Single Cells based on Function Using Lab on a Particle Technology
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

We have developed 3D-shaped hydrogel microparticle platforms to capture cells, as well as isolate and label their secretions. These “lab on a particle” systems enable sorting cells based on secreted products for the discovery of antibodies, the development of cell lines producing recombinant products, and the selection of functional cells for cell therapies. Each cell and its secreted products can be analyzed using widely available flow cytometers operating at up to a 1000 cells per second. I will discuss our latest results in sorting antigen-specific T cells and mesenchymal stem cells based on secreted cytokines, extracellular vesicles, and growth factors. Cells sorted based on these properties are intact, can regrow, and maintain the selected function over multiple population doublings, enabling cell therapy discovery and manufacturing workflows.