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Introduction to the Topics and Themes Addressed Across the Conference Tracks

The culmination of digital manufacturing is the seamless manufacture of a functional device from a digital design. Additive manufacturing is now used for making microfluidic chips, but microfluidics digital manufacturing is lagging, notably because functional systems depend on proprietary peripherals and a computer, and rely on generic, mass manufactured chips. Here I will discuss capillaric circuits (CCs) which are capillary-driven microfluidics that structurally encode simple algorithms of flow events into the circuit microarchitecture, and illustrate how application-specific CCs can be 3D printed using common, widely available stereolithography 3D printers. By encoding so-called microfluidic chain reactions, CCs can be programmed to execute step-by-step hundreds of sequential fluidic operations powered by a paper only, without electricity of peripheral connections. CCs have notably been used for automating an ELISA-on-a-chip for COVID19 antibody and antigen assays, and for the first microfluidic thrombin generation assay. Thanks to a new hydrophilic resin formulation, it is now possible to download a CC design, 3D-print it – which we demonstrate using ultra low-cost (US$300) LCD printers – clean it and use it within 30 minutes. These advances open the door for distributed and digital manufacturing of functional microfluidic CCs and systems by anyone, anywhere, anytime.

The integration of chemical functionalities in microfluidic devices is mostly accompanied by the combination of different materials. 3D printing has readily been proposed as manufacturing alternative for traditional approaches, in particular for small scale production. In particular in resin-based printers, however, the combination of different materials remains a challenge. This presentation focuses on the development of resins for digital light projection 3D printing of porous materials, and their integration into fluidic devices by resin exchange and using greyscale masks. Applications of the devices include phase separation, chemotaxis, electroextraction of DNA and the detection of iron from soil.

Circulating Tumor Cells (CTCs) are the holy grail of liquid biopsy biomarkers in oncology, however they are very rare, difficult to isolate and have not yet demonstrated clinical utility. Microfluidics has demonstrated numerous advantages for CTC isolation and characterization, with increased sensitivity and throughput, enabling their implementation in clinical routine. In this talk, we present our most recent work for the development of robust CTC assays, and their application in several clinical scenarios.

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Technological developments; such as ddPCR or optimized NGS, have greatly facilitated the tracking of circulating cell-free nucleic acids in body effluents. Several strategies can be designed to detect and characterize circulating tumor DNA (ctDNA) using ddPCR. Examples of such strategies will be presented that are based either cancer-specific methylation markers or DNA integrity evaluation. We will then illustrate the pertinence of these approaches for the detection and monitoring of ctDNA in plasma (so-called liquid biopsy) of patients with localized or advanced cancers. Results of several prospective clinical studies will be presented.

Nanofluidic devices offer promising and highly innovative approaches for analyzing single molecules and obtaining biophysical information that cannot be realized using microfluidics due to scaling issues. The ability to provide reliable, rapid, quantitative, and low-cost identification of single molecules will offer exciting new opportunities for a broad range of biomedical applications. We are developing dual in-plane nanopore sensors in plastics for the label-free detection and identification of single molecules. The hypothesis behind our nanopore sensor is, “individual molecules moving electrokinetically through a 2D nanotube will experience a time-of-flight (ToF) that are dependent upon their molecular identity.” In this presentation we will discuss the high rate manufacturing of a nanopore sensor with sub-5 nm in-plane nanopores using nano-injection molding from a cyclic olefin polymer (COP) plastic. The in-plane pores are situated at either end of a nanochannel (50 x 50 nm; 5 µm long) that generate current transient signals to detect and deduce the identity of the single molecule. The ToF is dependent on the apparent electrophoretic mobility of the molecule. The identity is determined from the ToF, the current transient amplitudes, and dwell times using multi-parameter Principle Component Analysis (PCA). We will show the ability to detect (detection efficiency ~100%) single ribonucleotide and deoxynucleotide monophosphates with identification accuracies exceeding 98%. Integrating the in-plane nanopore sensor with a solid-state nanoreactor results in a nanofluidic device that can be configured to provide molecular information from unamplified DNA/RNA targets with unprecedented capabilities. This will transform single-molecule processing to allow servicing a broad biomedical community for a wide range of applications, for example single-molecule sequencing.

Rapid and clinical scale isolation of extracellular vesicles (EV) from biofluids remains a major bottle neck in EV biomarker screening studies. Microfluidics has over the past years demonstrated several potential principles for EV isolation, however commonly suffering from limited capacity and/or sample throughput. In this perspective, acoustic nanoparticle trapping has emerged as an option for EV enrichment from biofluids where acoustic sound scattering, between microbeads and extra cellular vesicles, enrich EVs onto microbeads in a stationary cluster that is retained by the local acoustic field gradient. The technique has been used in investigations of EVs in blood plasma and urine. Traditionally this has been performed at a sample flow rate of ˜ 20 uL/min in a half wavelength acoustic standing wave trap, localized in a glass capillary, which when processing larger samples volumes, for increased biomarker sensitivity or when enriching EVs from dilute biofluids such as urine, suffer from extended processing times. To overcome this limitation we have developed an acoustic trapping unit, with ten standing wave nodes, that offers up to 40X increased trapping capacity and 25-40X increased sample processing flow rate. A typical sample flow rate of 500 uL/min enables rapid EV isolation and washing of milliliter volumes of urine as well as blood plasma in minutes prior to MS proteomic analysis. The proteome analysis of the acoustically enriched EV fraction displays clustering of proteins at elevated levels as compared to the input sample, suggesting a sub proteome specifically linked to the EVs. Furthermore, studies on EVs derived from pathogen activated platelets will be presented.

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Pre-analytics, or the manipulation and transformation of raw samples prior to their analysis, remains an important challenge for point-of-care devices. Demonstrating new sensing modalities on raw, rather than contrived samples, is sometimes considered as the ‘last frontier’. If done improperly, the pre-analytical phase can impact the quality of the sample analysis, and lead to diagnostic errors. This presentation will detail the design, production and use of simple, robust and modular devices for venous and capillary blood handling (covering blood transportation and metering, blood lysis, plasma separation and cell-free nucleic extraction) in microfluidic format. Our typical starting sample volume range spans from a few microliters to several milliliters and our devices have applications in point-of-care liquid biopsy workflows, from prenatal testing, drug-induced liver injury, and infectious disease diagnostic.

When starting a microfluidic project, many specifications are unknown and we as manufacturer challenge our customers a lot: Which specifications derived from the application requirements need to be met and controlled within certain tolerances in production? Not only but especially for applications requiring nanometer precision, it is of high importance to control the manufacturing processes like molding and bonding, but also set up proper characterization techniques to ensure continuous quality. We will show the realization and implementation using the example of Cytovale’s IntelliSep® chip having the need of microfluidic channels with nanometer precision for Sepsis Diagnostics.

Fluid manipulation on the micro-scale allows new applications in various fields including life sciences and diagnostics. Glass sometimes is the only applicable material due to limiting optical specifications or very harsh operating conditions. By harvesting the power of CMOS manufacturing technologies with non-CMOS compatible materials on glass, cost effective consumables for life sciences become reality in all volumes from prototyping to large scale manufacturing. At the overlap of semiconductor manufacturing technologies and microfluidics a multitude of added functionality can be incorporated into glass chips, enabling a new class of consumables for life sciences and diagnostic applications.

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Over the last 15 years, Kloé company developed a complete range of equipment dedicated to UV lithography applications, in perfect agreement with the microfabrication requirements in Microfluidics. Thus, Kloé company did the bet, in the early 2000, that the development of researches and industry in Microfluidics would rapidly grow. So that, over the same time, Kloé company continuously followed and exchanged with the Microfluidics community to first well understand and then anticipate its needs in terms of microfabrication techniques and performance, in order to enable fabricating from simple to more demanding microfluidic chips like Lab on a Chip/Organ on a Chip. Among a very large range of 12 different machines, covering from soft lithography / masking systems to very high resolution direct laser writers particularly suitable for fast prototyping, high aspect ratio as well as thick layers laser processing, Kloé introduces one of its latest innovations that is Dilase3D : a 3D-Printer specifically developed to meet the expectations for 3D-printing in Microfluidics. Typically elaborated from the specifications of researches in Microfluidics and Medical Sciences, that were looking for one tool enabling to both fabricate large volume pieces, but still with very high-resolution patterning capabilities, this equipment also demonstrated more recently its capability to combine different materials for the fabrication of one piece/object, that multiplies its capabilities to fabricate very demanding and ever more complex microchips/microstructures. This way, we ensure our partners to benefit from the one of the most performing and cost effective 3D-printing solution in that domain, in agreement with their expected level of performance and their available budget.

As 3D printing replaces traditional clean room manufacturing for microfluidic engineering applications, it’s becoming clear that this transition offers not only lower cost and faster design iterations, but also new opportunities for fluidic routing and control that are only possible due to the inherent three-dimensional nature of these systems. Over the past several years, we have developed design principles that take advantage of this three-dimensionality, as well as demonstrating several applications that benefit from this approach. At the core of these design principles is the modularity of microfluidic unit operations. In addition to designing prototypical unit operations such as mixers, splitters, flow focusers, droplet generators, thermal and optical sensors, and world-to-chip interfaces, we have developed systematic approaches to combining these modules into microfluidic circuits with predictable behaviors. This approach can be used to rapidly prototype complex microfludic operations by assembling physically distinct modules as well as to design monolithic microfluidic devices which can be printed in a single run. We have demonstrated the power of this approach by building several micro- and milifluidic systems for bio-analysis and bioprocess applications. These include systems for biomarker diagnostics, automated high-throughput affinity screening, and rapid manufacturing of vaccine lipid nanoparticles. We have also demonstrated how entire systems can be treated as modules, allowing for scaling of bioprocess production lines by massive parallelization.

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Chip-scale optofluidic devices have emerged as powerful, ultrasensitive sensors for molecular biomarkers. Maintaining outstanding performance in a point-of-care setting introduces new tradeoffs between cost, reliability, and performance. I will discuss the use of advanced signal processing methods that provide optimized information extraction in the presence of limited signal-to-noise ratios that are typically found outside a research lab. Examples include a new, ultrafast wavelet-based signal analysis algorithm, the use of machine learning and neural networks for multiplexing, and a new modulation technique that provides ultrawide dynamic range with a single method. In concert, these techniques allow for real-time, single molecule detection at the edge.

The Little Things Factory covers the whole portfolio to set up new functionalities for microfluidic systems in glass and we describe recent innovations in this field. The talk will introduce new possibilities to produce PLGA nanoparticles using flow reactors with high throughput and new flow reactors with catalysts in flow.

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Microfluidics have recently garnered considerable interest due to their unique benefits over conventional liquid handling systems and the tools they provide towards the development of lab-on-a-chip devices and organ-on-a-chip systems. Irrespectively of the specific application, the surface properties of microfluidic channels have to be carefully engineered to render them biocompatible and/or antifouling, customize their wettability or provide anchoring points for biomolecule tethering. Despite the significant progress made, surface (bio) functionalization remains a hard-to-navigate step in the fabrication process of microfluidics-based devices, as modification strategies are not only dependent on the basal material but also difficult to scale up at a low cost. Herein, an altogether different approach towards surface functionalization is presented that is based on the established toolbox of catechol-containing molecules as well as on the largely unexplored chemistry of aryl diazonium salts, both of which allow the highly specific yet universal, material-independent tailoring of the properties of microfluidic channels. The versatility of both chemical modification routes, developed within the H2020 NextGenMicrofluidics project, is showcased through their implementation into four different projects with distinct requirements, undertaken within the context of the Microfluidics Innovation Hub, a Single Entry Point where surface functionalization of microfluidics devices is offered as a service.

The presentation covers the activities of the HydroChip2 project (FFG No. 883914), in which the consortium targets on the realization of a point-of-care system for the detection of nucleic acids for the identification of periodontal pathogens.

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A microfluidic device consisting of an oscillating width channel was used to obtain the mechanical signature of red blood cells. We have demonstrated that by monitoring the shape and dynamics of cells exiting the last geometric restriction, we have managed to discriminate between blood samples from healthy donnors and patients with malaria, sickle cell disease and hereditary spherositosis.

We present a novel technique using double emulsion droplets produced with microfluidics that allows, for the first time, quantitative measurements of osmotic pressure both intra- and extra-cellularly within living embryonic tissues, including developing embryos.

The microfluidics innovation hub (MIH) acts as single entry point to the world’s largest platform for microfluidic services. In this presentation, we present a number of cutting-edge technologies in assay development and microfluidic manufacture in the context of on-going customer projects and demo cases.

This talk will discuss the microfluidics based approaches used in our lab to generate cell encapsulated microparticles and microcapsules. Particular focus will be on chemical gelation solidification technique and application of electric fields to control particle size.