SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

FLUIDOT: A Modular Microfluidic Platform for Single-Cell Study and Retrieval, with Applications in Drug Tolerance Screening and Antibody Mining.

Advancements in lab-on-a-chip technologies have revolutionized the single-cell analysis field. However, an accessible platform for in-depth screening and specific retrieval of single cells, which moreover enables studying diverse cell types and performing various downstream analyses, is still lacking. As a solution, FLUIDOT is introduced, a versatile microfluidic platform incorporating customizable microwells, optical tweezers and an interchangeable cell-retrieval system. Thanks to its smart microfluidic design, FLUIDOT is straightforward to fabricate and operate, rendering the technology widely accessible. The performance of FLUIDOT is validated and its versatility is subsequently demonstrated in two applications. First, drug tolerance in yeast cells is studied, resulting in the discovery of two treatment-tolerant populations. Second, B cells from convalescent COVID-19 patients are screened, leading to the discovery of highly affine, in vitro neutralizing monoclonal antibodies against SARS-CoV-2. Owing to its performance, flexibility, and accessibility, it is foreseen that FLUIDOT will enable phenotypic and genotypic analysis of diverse cell samples and thus elucidate unexplored biological questions.

Highly flexible and accurate serial picoinjection in droplets by combined pressure and flow rate control.

Picoinjection is a robust method for reagent addition into microfluidic droplets and has enabled the implementation of numerous multistep droplet assays. Although serial picoinjectors allow to screen many conditions in one run by injecting different combinations of reagents, their use is limited because it is complex to accurately control each injector independently. Here, we present a novel method for flexible, individual picoinjector control that allows to inject a predefined range of volumes by controlling the flow rate of the injector as well as turning off injection by setting the equilibrium pressure, which resulted in a stable interface of the injector liquid with the main microfluidic channel. Robust setting of the equilibrium pressure of an injector was achieved by applying accurate (R2 > 0.94) linear models between the injector and oil pressure in real-time. To illustrate the flexibility of this method, we performed several proof-of-concepts using 1, 2 or 3 picoinjectors loaded with fluorescent dyes. We successfully demonstrated picoinjection approaches using time-invariant settings, in which an injector setting was applied for prolonged times, and time-variant picoinjection, in which the injector settings were continuously varied in order to sweep the injected volumes, both resulting in monodisperse (CV < 3.4%) droplet libraries with different but reproducible fluorescent intensities. To illustrate the potential of the technology for fast compound concentration screenings, we studied the effect of a concentration range of a detergent on single-cell lysis. We anticipate that this robust and versatile methodology will make the serial picoinjection technology more accessible to researchers, allowing its wide implementation in numerous life science applications.

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells.

Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast.

Miniaturized single-cell technologies for monoclonal antibody discovery.

Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery.

Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes.

Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immuno-RCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.

Tuning the Surface Interactions between Single Cells and an OSTE+ Microwell Array for Enhanced Single Cell Manipulation.

Retrieving single cells of interest from an array of microwells for further off-chip analysis is crucial in numerous biological applications. To this end, several single cell manipulation strategies have been developed, including optical tweezers (OT). OT represent a unique approach for contactless cell retrieval, but their performance is often suboptimal due to nonspecific cell adhesion to the microwell surface. In this study, we focused on improving the surface chemistry of microwell arrays to ensure efficient single cell manipulation using OT. For this purpose, the surface of an off-stoichiometry thiol-ene-epoxy (OSTE+) microwell array was grafted with polyethylene glycol (PEG) molecules with different molecular weights: PEG 360, PEG 500, PEG 2000, and a PEG Mix (an equimolar ratio of PEG 500 and PEG 2000). Contact angle measurements showed that the PEG grafting process resulted in an increased surface energy, which was stable for at least 16 weeks. Next, cell adhesion of two cell types, baker's yeast (Saccharomyces cerevisiae) and human B cells, to surfaces treated with different PEGs was evaluated by registering the presence of cellular motion inside microwells and the efficiency of optical lifting of cells that display motion. Optimal results were obtained for surfaces grafted with PEG 2000 and PEG Mix, reaching an average fraction of cells with motion of over 93% and an average lifting efficiency of over 96% for both cell types. Upon the integration of this microwell array with a polydimethylsiloxane (PDMS) microfluidic channel, PEG Mix resulted in proper washing of non-seeded cells. We further demonstrated the wide applicability of the platform by manipulating non-responding yeast cells to antifungal treatment and B cells expressing surface IgG antibodies. The combination of the optimized microwell surface with continuous microfluidics results in a powerful and versatile platform, allowing high-throughput single cell studies and retrieval of target cells for off-chip analysis.

Reaction injection molding of hydrophilic-in-hydrophobic femtolitre-well arrays.

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.

Publisher Correction: Using high-amplitude and focused transcranial alternating current stimulation to entrain physiological tremor.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Using high-amplitude and focused transcranial alternating current stimulation to entrain physiological tremor.

Transcranial alternating current stimulation (tACS) is a noninvasive neuromodulation method that can entrain physiological tremor in healthy volunteers. We conducted two experiments to investigate the effectiveness of high-amplitude and focused tACS montages at entraining physiological tremor. Experiment 1 used saline-soaked sponge electrodes with an extra-cephalic return electrode and compared the effects of a motor (MC) and prefrontal cortex (PFC) electrode location. Average peak-amplitude was 1.925 mA. Experiment 2 used gel-filled cup-electrodes in a 4 × 1 focused montage and compared the effects of MC and occipital cortex (OC) tACS. Average peak-amplitude was 4.45 mA. Experiment 1 showed that unfocused MC and PFC tACS both produced phosphenes and significant phase entrainment. Experiment 2 showed that focused MC and OC tACS produced no phosphenes but only focused MC tACS caused significant phase entrainment. At the group level, tACS did not have a significant effect on tremor amplitude. However, with focused tACS there was a significant correlation between phase entrainment and tremor amplitude modulation: subjects with higher phase entrainment showed more tremor amplitude modulation. We conclude that: (1) focused montages allow for high-amplitude tACS without phosphenes and (2) high amplitude focused tACS can entrain physiological tremor.

Creasensor: SIMPLE technology for creatinine detection in plasma.

The lab-on-a-chip (LOC) field has witnessed an excess of new technology concepts, especially for the point-of-care (POC) applications. However, only few concepts reached the POC market often because of challenging integration with pumping and detection systems as well as with complex biological assays. Recently, a new technology termed SIMPLE was introduced as a promising POC platform due to its features of being self-powered, autonomous in liquid manipulations, cost-effective and amenable to mass production. In this paper, we improved the SIMPLE design and fabrication and demonstrated for the first time that the SIMPLE platform can be successfully integrated with biological assays by quantifying creatinine, biomarker for chronic kidney disease, in plasma samples. To validate the robustness of the SIMPLE technology, we integrated a SIMPLE-based microfluidic cartridge with colorimetric read-out system into the benchtop Creasensor. This allowed us to perform on-field validation of the Creasensor in a single-blind study with 16 plasma samples, showing excellent agreement between measured and spiked creatinine concentrations (ICC: 0.97). Moreover, the range of clinically relevant concentrations (0.76-20 mg/dL), the sample volume (5 µL) and time-to-result of only 5 min matched the Creasensor performance with both lab based and POC benchmark technologies. This study demonstrated for the first time outstanding robustness of the SIMPLE in supporting the implementation of biological assays. The SIMPLE flexibility in liquid manipulation and compatibility with different sample matrices opens up numerous opportunities for implementing more complex assays and expanding its POC applications portfolio.