Population assessments of infection and immunity status are critical for public health response to infectious disease. Most microfluidic tools are developed to assess one or the other - few assess both. This study introduces a multiplexed, fully automated digital microfluidic (DMF) platform designed to detect viral protein as a proxy for infection status and host IgG and IgA antibodies as a marker for immunity status. SARS-CoV-2 and patient saliva were used as a model system to evaluate the concept. Specifically, the infection assay relied on nanobody-based capture and detection agents specific to SARS-CoV-2 trimeric spike protein, with a limit of detection (LOD) of 3.8 ng mL-1 in saliva. And the immunity relied on monoclonal antibodies for host IgG and IgA specific to SARS-CoV-2 spike S1 domain, with LODs of 4.8 ng mL-1 and 13.3 ng mL-1 in saliva, respectively. Clinical validation in saliva samples from human subjects experiencing symptoms (n = 14) showed strong correlation with PCR and commercial ELISA, achieving 100% sensitivity and 100% specificity for infection detection and 100% sensitivity with 91.7% and 90.9% specificity for host IgG and IgA, respectively. These results highlight potential applications for the new system as a portable diagnostic tool for outbreak surveillance and public health management, as a step toward preparing for the next global pandemic.

Micromachines capable of performing diverse mechanical tasks in complex and constrained microenvironments are of great interest. Despite important milestones in this pursuit, until now, micromachines are confined to actuation within a single 2D plane due to the challenges of transferring motion across different planes in limited space. Here, a breakthrough method is presented to overcome this limitation: multi-component micromachines that facilitate 3D motion transfer across different planes. These light-driven 3D micromachines, fabricated using standard photolithography combined with direct laser writing, are assembled and actuated via programmable light patterns within an optoelectronic tweezers system. Utilizing charge-induced repulsion and dielectrophoretic levitation effects, the micromachines enable highly efficient mechanical rotation and effective inter-component motion transfer. Through this work, fascinating patterns of similarities are unveiled for the new microscale 3D systems when compared with the macro-scale world in which they live, paving the way for the development of micromechanical devices and microsystems with ever increasing functionality and versatility.

We report methods that improve the manipulation of magnetic beads using digital microfluidics (DMF) that can enhance the performance of single molecule array (Simoa) digital protein assays in miniaturized analytical systems. Despite significant clinical and biomedical applications for digital protein detection, the development of miniaturized Simoa systems has been limited by the requirements for use of large sample volumes (∼100 μL) and low numbers of beads (∼5000) for high sensitivity tests. To address these challenges, we improved the integration of DMF with Simoa-based assays by developing strategies for loading mixtures of sample and beads into DMF networks using methods relying on either virtual channels or small liquid segments that were applied either in parallel or in a stepwise manner. We have also demonstrated a dedicated densifying electrode technique that captures low numbers of beads within a droplet, allowing high bead retention with minimal residual volumes of liquid. Based on these improvements, we optimized the front-end assay processing of beads using DMF and demonstrated a method to detect tumor necrosis factor α (TNF-α) by Simoa that showed equivalent performance to a microtitre plate assay. The new strategies described here form a step toward integrating DMF and Simoa for a wide range of applications.

Digitalizing the signals generated from single protein molecules has significantly improved the sensitivity of immunoassays compared to traditional analog "bulk" measurements. The single molecule array (Simoa) technology, for instance, leverages counting of single molecules on magnetic beads to detect low-abundance proteins in biofluids. While existing digital detection platforms are ultra-sensitive, they typically require compartmentalization and complex and bulky analysis equipment, limiting their applicability in resource-limited settings. Here, we introduce a compartmentalization-free digital detection technique, that allows for much more straightforward detection analysis. We applied this method to a model assay for detecting the SARS-CoV-2 spike protein and compared its performance to alternative techniques. We optimized the new method for digital microfluidics and present preliminary results using an automated system to analyze undiluted human saliva samples, with imaging performed on a portable optical system. We propose that future iterations of the scheme introduced here have the potential to enable a wide range of applications beyond the laboratory.

We describe an approach to enhancing microfluidic mixing by generating acoustic microstreaming flows around microposts in a microfluidic device. Specifically, we synthesize microposts with various cross-sectional shapes (i.e., circles, triangles, and stars) using photocrosslinkable polymers, allowing for precise control over their geometry. We also ensure unobstructed micropost vibration via carefully designed gaps between the microposts and the channel ceiling. Experimental findings reveal that the shape of microposts is critical in influencing microstreaming patterns and mixing efficiency. Circular microposts generate semi-symmetrical circular vortices, resulting in superior mixing performance (86.7

Long-term memory (LTM) formation relies on de novo protein synthesis; however, the full complement of proteins crucial to LTM formation remains unknown in any system. Using an aversive operant conditioning model of aerial respiratory behavior in the pond snail mollusk, Lymnaea stagnalis (L. stagnalis), we conducted a transcriptome-guided proteomic analysis on the central nervous system (CNS) of LTM, no LTM, and control animals. We identified 366 differentially expressed proteins linked to LTM formation, with 88 upregulated and 36 downregulated in LTM compared to both no LTM and controls. Functional annotation highlighted the importance of balancing protein synthesis and degradation for LTM, as indicated by the upregulation of proteins involved in proteasome activity and translation initiation, including EIF2D, mRNA levels of which were confirmed to be upregulated by conditioning and implicated nuclear factor Y as a potential regulator of LTM-related transcription in this model. This study represents the first transcriptome-guided proteomic analysis of LTM formation ability in this model and lays the groundwork for discovering orthologous proteins critical to LTM in mammals.

Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.

Microproteomics, the profiling of protein expressions in small cell populations or individual cells, is essential for understanding complex biological systems. However, sample loss and insufficient sensitivity of analytical techniques pose severe challenges to this field. Microfluidics, particularly droplet-based microfluidics, provides an ideal approach by enabling miniaturized and integrated workflows to process samples and offers several advantages, including reduced sample loss, low reagent consumption, faster reaction times, and improved throughput. Droplet-based microfluidics manipulates droplets of fluids to function as discrete reaction units, enabling complex chemical reactions and biological workflows in a miniaturized setting. This article discusses a variety of on-chip functions of droplet-based microfluidics, including cell sorting, cell culture, and sample processing. We then highlight recent advances in the mass spectrometry (MS)-based analysis of single cells using droplet-based microfluidic platforms, including digital microfluidics (DMF). Finally, we review the integrated DMF–MS systems that enable automated and parallel proteomic profiling of single cells with high sensitivity and discuss the applications of the technology and its future perspectives.

Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO ( tissue- digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5 . Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.

AbstractForensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short‐tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non‐sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post‐coitus (PC), the new method identifies single‐source (male) STR profiles. When applied to samples collected 24–72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre‐processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.

Skeletal muscle regeneration is mediated by resident muscle stem cells that produce progeny to repair or recreate muscle. Critical to this function is the ability to transition between states of proliferation and quiescence. This balancing act is disrupted with age, leading to eroded regenerative capacity. Notwistanding, mechanisms by which the regenerating niche directs MuSCs return to the dormant state are largely unknown. Since single-cell RNAseq methods exclude the analysis of multinucleated cells, we generated single-nuclei RNAseq datasets of regenerating muscle to capture the full breadth of myogenic progression. With this, we uncovered new transition states between differentiating myocytes and syncytial multinucleated cells. Using cell communication inference tools, we highlighted receptor-ligand interactions between MuSCs and fusing nuclei. We leveraged a bespoke biomimetic 3D niche that induces MuSC quiescence, to filter the predicted interactions using a Cas9-based functional genomics approach. We found the proteoglycan Perlecan (Hspg2) promotes MuSC re-quiescence. Hspg2 silencing in vivo , during muscle regeneration, induced an aging-like phenotype and perturbed MuSC re-quiescence. Notably, the temporal profile and overall levels of Perlecan were altered with age. Exogenous supplementation with Endorepellin (truncated Perlecan) rescued MuSC decline following aged muscle regeneration, offering a new therapeutic target. Thus, coupling in silico predictions with a 3D in vitro assay followed by in vivo investigations revealed a previously inaccessible window of biology; that spatiotemporal coordination of MuSC re-quiescence is directed by fusing myonuclei. ### Competing Interest Statement The authors have declared no competing interest.

In recent years, monoclonal antibodies (mAbs) have become a powerful tool in the treatment of human diseases. Currently, over 100 mAbs have received approval for therapeutic use in the US, with wide-ranging applications from cancer to infectious diseases. The predominant method of producing antibodies for therapeutics involves expression in mammalian cell lines. In the mAb production process, significant optimization is typically done to maximize antibody titres from cells grown in bioreactors. Therefore, systems that can miniaturize and automate cell line testing (e.g., viability and antibody production assays) are valuable in reducing therapeutic mAb development costs. Here we present a novel platform for cell line optimization for mAb production using digital microfluidics. The platform enables testing of cell culture samples in 6-8 μL droplets with semi-automated viability, media pH, and antibody production assays. This system provides a unique bridge between cell growth and productivity metrics, while minimizing culture volume requirements for daily testing. We propose that this technology and its future iterations has the potential to help reduce the time-to-market and development costs of antibody-producing cell lines.

Toxicity testing is currently undergoing a paradigm shift from examining apical end points such as death, to monitoring sub-lethal toxicity in vivo. In vivo nuclear magnetic resonance (NMR) spectroscopy is a key platform in this endeavor. A proof-of-principle study is presented which directly interfaces NMR with digital microfluidics (DMF). DMF is a "lab on a chip" method allowing for the movement, mixing, splitting, and dispensing of μL-sized droplets. The goal is for DMF to supply oxygenated water to keep the organisms alive while NMR detects metabolomic changes. Here, both vertical and horizontal NMR coil configurations are compared. While a horizontal configuration is ideal for DMF, NMR performance was found to be sub-par and instead, a vertical-optimized single-sided stripline showed most promise. In this configuration, three organisms were monitored in vivo using 1H-13C 2D NMR. Without support from DMF droplet exchange, the organisms quickly showed signs of anoxic stress; however, with droplet exchange, this was completely suppressed. The results demonstrate that DMF can be used to maintain living organisms and holds potential for automated exposures in future. However, due to numerous limitations of vertically orientated DMF, along with space limitations in standard bore NMR spectrometers, we recommend future development be performed using a horizontal (MRI style) magnet which would eliminate practically all the drawbacks identified here.

Single-cell proteomics is emerging as an important subfield in the proteomics and mass spectrometry communities, with potential to reshape our understanding of cell development, cell differentiation, disease diagnosis, and the development of new therapies. Compared with significant advancements in the "hardware" that is used in single-cell proteomics, there has been little work comparing the effects of using different "software" packages to analyze single-cell proteomics datasets. To this end, seven popular proteomics programs were compared here, applying them to search three single-cell proteomics datasets generated by three different platforms. The results suggest that MSGF+, MSFragger, and Proteome Discoverer are generally more efficient in maximizing protein identifications, that MaxQuant is better suited for the identification of low-abundance proteins, that MSFragger is superior in elucidating peptide modifications, and that Mascot and X!Tandem are better for analyzing long peptides. Furthermore, an experiment with different loading amounts was carried out to investigate changes in identification results and to explore areas in which single-cell proteomics data analysis may be improved in the future. We propose that this comparative study may provide insight for experts and beginners alike operating in the emerging subfield of single-cell proteomics.

On November 18-19, 2019, the Immunity of Canadians and Risk of Epidemics (iCARE) Network convened a workshop in Toronto, Ontario, Canada. The objectives of the workshop were to raise the profile of sero-epidemiology in Canada, discuss best practice and methodological innovations, and strategize on the future direction of sero-epidemiology work in Canada. In this conference report, we describe the presentations and discussions from the workshop, and comment on the impact of the COVID-19 pandemic on serosurveillance initiatives, both in Canada and abroad.

The identification of tumor-specific antigens (TSAs) is critical for developing effective cancer immunother-apies. Mass spectrometry (MS)-based immunopeptidomics has emerged as a powerful tool for identifying TSAs as physical molecules. However, current immunopeptidomics platforms face challenges in measuring low-abundance TSAs in a precise, sensitive, and reproducible manner from small needle-tissue biopsies (<1 mg). Inspired by recent advances in single-cell proteomics, microfluidics technology offers a promising solution to these limitations by providing improved isolation of human leukocyte antigen (HLA)-associated peptides with higher sensitivity. In this context, we highlight the challenges in sample preparation and the rationale for developing microfluidics technology in immunopeptidomics. Additionally, we provide an over-view of promising microfluidic methods, including microchip pillar arrays, valved-based systems, droplet microfluidics, and digital microfluidics, and discuss the latest research on their application in MS-based im-munopeptidomics and single-cell proteomics.

There is great enthusiasm for using loop-mediated isothermal amplification (LAMP) in point-of-care nucleic acid amplification tests (POC NAATs), as an alternative to PCR. While isothermal amplification techniques like LAMP eliminate the need for rapid temperature cycling in a portable format, these systems are still plagued by requirements for dedicated optical detection apparatus for analysis and manual off-chip sample processing. Here, we developed a new microfluidic system for LAMP-based POC NAATs to address these limitations. The new system combines digital microfluidics (DMF) with distance-based detection (DBD) for direct signal readout. This is the first report of the use of (i) LAMP or (ii) DMF with DBD - thus, we describe a number of characterization steps taken to determine optimal combinations of reagents, materials, and processes for reliable operation. For example, DBD was found to be quite sensitive to background signals from low molecular weight LAMP products; thus, a Capto™ adhere bead-based clean-up procedure was developed to isolate the desirable high-molecular-weight products for analysis. The new method was validated by application to detection of SARS-CoV-2 in saliva. The method was able to distinguish between saliva containing no virus, saliva containing a low viral load (104 genome copies per mL), and saliva containing a high viral load (108 copies per mL), all in an automated system that does not require detection apparatus for analysis. We propose that the combination of DMF with distance-based detection may be a powerful one for implementing a variety of POC NAATs or for other applications in the future.

Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Dimethyl labeling was integrated into the pipeline to allow for relative quantification of the trace samples at the nanogram level, and the new pipeline was applied to evaluating cancer cell lines and cancer tissue samples. Several known proteins (including HSP90AB1, HSPB1, LDHA, ENO1, PGK1, KRT18, and AKR1C2) and pathways were observed between model breast cancer cell lines related to hormone response, cell metabolism, and cell morphology. Furthermore, differentially quantified proteins (such as PGS2, UGDH, ASPN, LUM, COEA1, and PRELP) were found in comparisons of healthy and cancer breast tissues, suggesting potential utility of the All-in-One pipeline for the emerging application of proteomic cancer sub-typing. In sum, the All-in-One pipeline represents a powerful new tool for automated proteome processing and analysis, with the potential to be useful for evaluating mass-limited samples for a wide range of applications.

Steering micromotors is important for using them in practical applications and as model systems for active matter. This functionality often requires magnetic materials in the micromotor, taxis behavior of the micromotor, or the use of specifically designed physical boundaries. Here, we develop an optoelectronic strategy that steers micromotors with programmable light patterns. In this strategy, light illumination turns hydrogenated amorphous silicon conductive, generating local electric field maxima at the edge of the light pattern that attracts micromotors via positive dielectrophoresis. As an example, metallo-dielectric Janus microspheres that self-propelled under alternating current electric fields were steered by static light patterns along customized paths and through complex microstructures. Their long-term directionality was also rectified by ratchet-shaped light patterns. Furthermore, dynamic light patterns that varied in space and time enabled more advanced motion controls such as multiple motion modes, parallel control of multiple micromotors, and the collection and transport of motor swarms. This optoelectronic steering strategy is highly versatile and compatible with a variety of micromotors, and thus it possesses the potential for their programmable control in complex environments.