Robust Double Emulsions for Multicolor Fluorescence-Activated Cell Sorting.

Cell-cell interactions are essential for the proper functioning of multicellular organisms. For example, T cells interact with antigen-presenting cells (APCs) through specific T-cell receptor (TCR)-antigen interactions during an immune response. Fluorescence-activated droplet sorting (FADS) is a high-throughput technique for efficiently screening cellular interaction events. Unfortunately, current droplet sorting instruments have significant limitations, most notably related to analytical throughput and complex operation. In contrast, commercial fluorescence-activated cell sorters offer superior speed, sensitivity, and multiplexing capabilities, although their use as droplet sorters is poorly defined and underutilized. Herein, we present a universally applicable and simple-to-implement workflow for generating double emulsions and performing multicolor cell sorting using a commercial FACS instrument. This workflow achieves a double emulsion detection rate exceeding 90%, enabling multicellular encapsulation and high-throughput immune cell activation sorting for the first time. We anticipate that the presented droplet sorting strategy will benefit cell biology laboratories by providing access to an advanced microfluidic toolbox with minimal effort and cost investment.

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Actomyosin-mediated apical constriction promotes physiological germ cell death in C. elegans.

Germ cell apoptosis in Caenorhabditis elegans hermaphrodites is a physiological process eliminating around 60% of all cells in meiotic prophase to maintain tissue homeostasis. In contrast to programmed cell death in the C. elegans soma, the selection of germ cells undergoing apoptosis is stochastic. By live-tracking individual germ cells at the pachytene stage, we found that germ cells smaller than their neighbors are selectively eliminated through apoptosis before differentiating into oocytes. Thus, cell size is a strong predictor of physiological germ cell death. The RAS/MAPK and ECT/RHO/ROCK pathways together regulate germ cell size by controlling actomyosin constriction at the apical rachis bridges, which are cellular openings connecting the syncytial germ cells to a shared cytoplasmic core. Enhancing apical constriction reduces germ cell size and increases the rate of cell death while inhibiting the actomyosin network in the germ cells prevents their death. We propose that actomyosin contractility at the rachis bridges of the syncytial germ cells amplifies intrinsic disparities in cell size. Through this mechanism, the animals can adjust the balance between physiological germ cell death and oocyte differentiation.

Quantitative reagent monitoring in paper-based electrochemical rapid diagnostic tests.

Paper-based rapid diagnostic tests (RDTs) are an essential component of modern healthcare, particularly for the management of infectious diseases. Despite their utility, these capillary-driven RDTs are compromised by high failure rates, primarily caused by user error. This limits their utility in complex assays that require multiple user operations. Here, we demonstrate how this issue can be directly addressed through continuous electrochemical monitoring of reagent flow inside an RDT using embedded graphenized electrodes. Our method relies on applying short voltage pulses and measuring variations in capacitive discharge currents to precisely determine the flow times of injected samples and reagents. This information is reported to the user, guiding them through the testing process, highlighting failure cases and ultimately decreasing errors. Significantly, the same electrodes can be used to quantify electrochemical signals from immunoassays, providing an integrated solution for both monitoring assays and reporting results. We demonstrate the applicability of this approach in a serology test for the detection of anti-SARS-CoV-2 IgG in clinical serum samples. This method paves the way towards "smart" RDTs able to continuously monitor the testing process and improve the robustness of point-of-care diagnostics.

Directed evolution of material-producing microorganisms.

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials.

FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Site-directed conjugation of single-stranded DNA to affinity proteins: quantifying the importance of conjugation strategy.

Affinity protein-oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein-oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein-oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein-oligonucleotide conjugates.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

Electrochemical Paper-Based Microfluidics: Harnessing Capillary Flow for Advanced Diagnostics.

Electrochemical paper-based microfluidics has attracted much attention due to the promise of transforming point-of-care diagnostics by facilitating quantitative analysis with low-cost and portable analyzers. Such devices harness capillary flow to transport samples and reagents, enabling bioassays to be executed passively. Despite exciting demonstrations of capillary-driven electrochemical tests, conventional methods for fabricating electrodes on paper impede capillary flow, limit fluidic pathways, and constrain accessible device architectures. This account reviews recent developments in paper-based electroanalytical devices and offers perspective by revisiting key milestones in lateral flow tests and paper-based microfluidics engineering. The study highlights the benefits associated with electrochemical sensing and discusses how the detection modality can be leveraged to unlock novel functionalities. Particular focus is given to electrofluidic platforms that embed electrodes into paper for enhanced biosensing applications. Together, these innovations pave the way for diagnostic technologies that offer portability, quantitative analysis, and seamless integration with digital healthcare, all without compromising the simplicity of commercially available rapid diagnostic tests.

Granular Biomaterials as Bioactive Sponges for the Sequestration and Release of Signaling Molecules.

A major challenge for the regeneration of chronic wounds is an underlying dysregulation of signaling molecules, including inflammatory cytokines and growth factors. To address this, it is proposed to use granular biomaterials composed of jammed microgels, to enable the rapid uptake and delivery of biomolecules, and provide a strategy to locally sequester and release biomolecules. Sequestration assays on model biomolecules of different sizes demonstrate that granular hydrogels exhibit faster transport than comparable bulk hydrogels due to enhanced surface area and decreased diffusion lengths. To demonstrate the potential of modular granular hydrogels to modulate local biomolecule concentrations, microgel scaffolds are engineered that can simultaneously sequester excess pro-inflammatory factors and release pro-healing factors. To target specific biomolecules, microgels are functionalized with affinity ligands that bind either to interleukin 6 (IL-6) or to vascular endothelial growth factor A (VEGF-A). Finally, disparate microgels are combined into a single granular biomaterial for simultaneous sequestration of IL-6 and release of VEGF-A. Overall, the potential of modular granular hydrogels is demonstrated to locally tailor the relative concentrations of pro- and anti-inflammatory factors.

A High Efficiency, Low Resistance Antibacterial Filter Formed by Dopamine-Mediated In Situ Deposition of Silver onto Glass Fibers.

The coating of filter media with silver is typically achieved by chemical deposition and aerosol processes. Whilst useful, such approaches struggle to provide uniform coating and are prone to blockage. To address these issues, an in situ method for coating glass fibers is presented via the dopamine-mediated electroless metallization method, yielding filters with low air resistance and excellent antibacterial performance. It is found that the filtration efficiency of the filters is between 94 and 97% and much higher than that of silver-coated filters produced using conventional dipping methods (85%). Additionally, measured pressure drops ranged between 100 and 150 Pa, which are lower than those associated with dipped filters (171.1 Pa). Survival rates of Escherichia coli and Bacillus subtilis bacteria exposed to the filters decreased to 0 and 15.7%±1.49, respectively after 2 h, with no bacteria surviving after 6 h. In contrast, survival rates of E. coli and B. subtilis bacteria on the uncoated filters are 92.5% and 89.5% after 6 h. Taken together, these results confirm that the in situ deposition of silver onto fiber surfaces effectively reduces pore clogging, yielding low air resistance filters that can be applied for microbial filtration and inhibition in a range of environments.

Droplet-based microfluidics and enzyme evolution.

Enzymes are widely used as catalysts in the chemical and pharmaceutical industries. While successful in many situations, they must usually be adapted to operate efficiently under nonnatural conditions. Enzyme engineering allows the creation of novel enzymes that are stable at elevated temperatures or have higher activities and selectivities. Current enzyme engineering techniques require the production and testing of enzyme variant libraries to identify members with desired attributes. Unfortunately, traditional screening methods cannot screen such large mutagenesis libraries in a robust and timely manner. Droplet-based microfluidic systems can produce, process, and sort picoliter droplets at kilohertz rates and have emerged as powerful tools for library screening and thus enzyme engineering. We describe how droplet-based microfluidics has been used to advance directed evolution.

Monitoring Aqueous Sucrose Solutions Using Droplet Microfluidics: Ice Nucleation, Growth, Glass Transition, and Melting.

Freezing and freeze-drying processes are commonly used to extend the shelf life of drug products and to ensure their safety and efficacy upon use. When designing a freezing process, it is beneficial to characterize multiple physicochemical properties of the formulation, such as nucleation rate, crystal growth rate, temperature and concentration of the maximally freeze-concentrated solution, and melting point. Differential scanning calorimetry has predominantly been used in this context but does have practical limitations and is unable to quantify the kinetics of crystal growth and nucleation. In this work, we introduce a microfluidic technique capable of quantifying the properties of interest and use it to investigate aqueous sucrose solutions of varying concentration. Three freeze-thaw cycles were performed on droplets with 75-µm diameters at cooling and warming rates of 1 °C/min. During each cycle, the visual appearance of the droplets was optically monitored as they experienced nucleation, crystal growth, formation of the maximally freeze-concentrated solution, and melting. Nucleation and crystal growth manifested as increases in droplet brightness during the cooling phase. Heating was associated with a further increase as the temperature associated with the maximally freeze-concentrated solution was approached. Heating beyond the melting point corresponded to a decrease in brightness. Comparison with the literature confirmed the accuracy of the new technique while offering new visual data on the maximally freeze-concentrated solution. Thus, the microfluidic technique presented here may serve as a complement to differential scanning calorimetry in the context of freezing and freeze-drying. In the future, it could be applied to a plethora of mixtures that undergo such processing, whether in pharmaceutics, food production, or beyond.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

Ultrahigh-Throughput, Real-Time Flow Cytometry for Rare Cell Quantification from Whole Blood.

We present an ultrahigh-throughput, real-time fluorescence cytometer comprising a viscoelastic microfluidic system and a complementary metal-oxide-semiconductor (CMOS) linear image sensor-based detection system. The flow cytometer allows for real-time quantification of a variety of fluorescence species, including micrometer-sized particles and cells, at analytical throughputs in excess of 400,000 species per second. The platform integrates a custom C++ control program and graphical user interface (GUI) to allow for the processing of raw signals, adjustment of processing parameters, and display of fluorescence intensity histograms in real time. To demonstrate the efficacy of the platform for rare event detection and its utility as a basic clinical tool, we measure and quantify patient-derived circulating tumor cells (CTCs) in peripheral blood, realizing that detection has a sensitivity of 6 CTCs per million blood cells (0.000006%) with a volumetric throughput of over 3 mL/min.

Correction: Real-time, smartphone-based processing of lateral flow assays for early failure detection and rapid testing workflows.

[This corrects the article DOI: 10.1039/D2SD00197G.].

Transforming Nanomaterial Synthesis with Flow Chemistry.

Microfluidic methods for the synthesis of nanomaterials allow the generation of high-quality products with outstanding structural, electronic and optical properties. At a fundamental level, this is engendered by the ability to control both heat and mass transfer in a rapid and precise manner, but also by the facile integration of in-line characterization tools and machine learning algorithms. Such integrated platforms provide for exquisite control over material properties during synthesis, accelerate the optimization of electronic and optical properties and bestow new insights into the optoelectronic properties of nanomaterials. Herein, we present a brief perspective on the role that microfluidic technologies can play in nanomaterial synthesis, with a particular focus on recent studies that incorporate in-line optical characterization and machine learning. We also consider the importance and challenges associated with integrating additional functional components within experimental workflows and the upscaling of microfluidic platforms for production of industrial-scale quantities of nanomaterials.

On-site airborne pathogen detection for infection risk mitigation.

Human-infecting pathogens that transmit through the air pose a significant threat to public health. As a prominent instance, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic has affected the world in an unprecedented manner over the past few years. Despite the dissipating pandemic gloom, the lessons we have learned in dealing with pathogen-laden aerosols should be thoroughly reviewed because the airborne transmission risk may have been grossly underestimated. From a bioanalytical chemistry perspective, on-site airborne pathogen detection can be an effective non-pharmaceutic intervention (NPI) strategy, with on-site airborne pathogen detection and early-stage infection risk evaluation reducing the spread of disease and enabling life-saving decisions to be made. In light of this, we summarize the recent advances in highly efficient pathogen-laden aerosol sampling approaches, bioanalytical sensing technologies, and the prospects for airborne pathogen exposure measurement and evidence-based transmission interventions. We also discuss open challenges facing general bioaerosols detection, such as handling complex aerosol samples, improving sensitivity for airborne pathogen quantification, and establishing a risk assessment system with high spatiotemporal resolution for mitigating airborne transmission risks. This review provides a multidisciplinary outlook for future opportunities to improve the on-site airborne pathogen detection techniques, thereby enhancing the preparedness for more on-site bioaerosols measurement scenarios, such as monitoring high-risk pathogens on airplanes, weaponized pathogen aerosols, influenza variants at the workplace, and pollutant correlated with sick building syndromes.

Direct isolation of small extracellular vesicles from human blood using viscoelastic microfluidics.

Small extracellular vesicles (sEVs; <200 nm) that contain lipids, nucleic acids, and proteins are considered promising biomarkers for a wide variety of diseases. Conventional methods for sEV isolation from blood are incompatible with routine clinical workflows, significantly hampering the utilization of blood-derived sEVs in clinical settings. Here, we present a simple, viscoelastic-based microfluidic platform for label-free isolation of sEVs from human blood. The separation performance of the device is assessed by isolating fluorescent sEVs from whole blood, demonstrating purities and recovery rates of over 97 and 87%, respectively. Significantly, our viscoelastic-based microfluidic method also provides for a remarkable increase in sEV yield compared to gold-standard ultracentrifugation, with proteomic profiles of blood-derived sEVs purified by both methods showing similar protein compositions. To demonstrate the clinical utility of the approach, we isolate sEVs from blood samples of 20 patients with cancer and 20 healthy donors, demonstrating that elevated sEV concentrations can be observed in blood derived from patients with cancer.