Sealable capped nanovials for high-throughput screening of cell growth and function.

Millions of modular nanoliter-scale compartments that isolate functionally rich single-cell and cell-to-cell communication data can scale biological discovery for the age of AI. Here, we introduce capped nanovials-suspendable, sealable microscale compartments formed by the docking of hydrogel capping particles into bowl-shaped nanovials-as a versatile system for culturing, analyzing, and sorting single cells and small colonies. This two-particle architecture enables localized confinement of cells and secreted products while maintaining compatibility with standard laboratory workflows such as wash and reagent exchange steps, fluorescence microscopy, and flow cytometry.

Screening of a pooled library of chimeric antigen receptor T cells based on secretory function.

Chimeric antigen receptor (CAR) T cell therapies have shown promise in treating hematologic malignancies, but challenges remain due to immune suppression, antigen heterogeneity, and insufficient functional screening platforms. Here, we present a modular nanovial-based platform for high-throughput, single-cell functional screening of pooled CAR T cell libraries. Nanovials, hydrogel microparticles with nanoliter-scale cavities, were functionalized with recombinant HER2 antigen and cytokine-capture antibodies to simulate antigen-presenting cells and capture secreted interferon-γ (IFNγ).

Functional screening of antibody-secreting cells by co-culture with reporter T cells using PicoShells.

Monoclonal antibodies (mAbs) are a growing class of therapeutics known for their high specificity and diverse functional mechanisms, including agonism and antagonism. Although microwell array technologies and droplet microfluidics are employed to pair antibody-secreting cells (ASCs) with target cells for therapeutic mAb discovery, existing methods suffer from limited throughput or inadequate functional assessment. To address these limitations, we applied PicoShells, hollow media-permeable hydrogel microparticles, to evaluate mAb function by co-culturing hybridomas with reporter cells for 24 hours.

Specific Stimulation and Multiplex Secretion Analysis of T Cells Using a Suspendable Hydrogel Platform Enables Immunophenotyping at the Single-Cell Scale.

The ability to characterize and separate cells based on their surface marker expression profiles using flow cytometry revolutionized our understanding of the immune system at the level of single cells. However, the use of surface protein quantification to functionally evaluate T cells following stimulation fails to capture important outcomes of T cell activation, most prominently, cytokine secretion. This key limitation hinders elucidation of the mechanistic correlates of T cell function, which in turn limits our ability to design effective cell therapies for diseases such as cancer and autoimmune disorders.

Linking Epitope-Specific T-Cell Receptors to IFNγ Secretion Using Nanovial Technology.

To link IFNγ-secretion levels of epitope-specific T-cells with their TCRαβ, we coated nanovials with pHLA-I to capture and activate epitope-specific T-cells and their secreted IFNγ, followed by index-sorting and TCRαβ sequencing. We demonstrate that nanovials are a promising tool to link single epitope-specific TCRαβ clonotypes to the cell's functional properties.

Paper-Based Vertical Flow Assays for in Vitro Diagnostics and Environmental Monitoring.

Microfluidic paper-based analytical devices (μPADs) are powerful tools for diagnostic and environmental monitoring. Being affordable and portable, μPADs enable rapid detection of small molecules, heavy metals, and biomolecules, thereby decentralizing diagnostics and expanding biosensor accessibility. However, the reliance on two-dimensional fluid flow restricts the utility of conventional μPADs, presenting challenges for applications that require simultaneous multibiomarker analysis from a single sample.

Machine learning in point-of-care testing: innovations, challenges, and opportunities.

The landscape of diagnostic testing is undergoing a significant transformation, driven by the integration of artificial intelligence (AI) and machine learning (ML) into decentralized, rapid, and accessible sensor platforms for point-of-care testing (POCT). The COVID-19 pandemic has accelerated the shift from centralized laboratory testing but also catalyzed the development of next-generation POCT platforms that leverage ML to enhance the accuracy, sensitivity, and overall efficiency of point-of-care sensors. This Perspective explores how ML is being embedded into various POCT modalities, including lateral flow assays, vertical flow assays, nucleic acid amplification tests, and imaging-based sensors, illustrating their impact through different applications.

Linking single-cell transcriptomes with secretion using SEC-seq.

Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions-from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown.

Deep Learning-Enhanced Chemiluminescence Vertical Flow Assay for High-Sensitivity Cardiac Troponin I Testing.

Democratizing biomarker testing at the point-of-care requires innovations that match laboratory-grade sensitivity and precision in an accessible format. Here, high-sensitivity detection of cardiac troponin I (cTnI) is demonstrated through innovations in chemiluminescence-based sensing, imaging, and deep learning-driven analysis. This chemiluminescence vertical flow assay (CL-VFA) enables rapid, low-cost, and precise quantification of cTnI, a key cardiac protein for assessing heart muscle damage and myocardial infarction.

Multi-reactive hydrogel nanovials for temporal control of secretion capture from antibody-secreting cells.

Antibody discovery can benefit from techniques to screen antibody-secreting cells (ASCs) at scale for the binding and functionality of a diverse set of secreted antibodies. Previously, we demonstrated the use of cavity-containing hydrogel microparticles (nanovials) coated with a single affinity agent, biotin, to capture and identify ASCs secreting antibodies against a recombinant antigen bound to the nanovial through biotin-streptavidin linkages. However, rapidly secreted antibodies from unbound cells or cells in adjacent nanovials can cause crosstalk leading to background signal.

Tunable Picoliter-Scale Dropicle Formation Using Amphiphilic Microparticles with Patterned Hydrophilic Patches.

Microparticle-templated droplets or dropicles have recently gained interest in the fields of diagnostic immunoassays, single-cell analysis, and digital molecular biology. Amphiphilic particles have been shown to spontaneously capture aqueous droplets within their cavities upon mixing with an immiscible oil phase, where each particle templates a single droplet. Here, an amphiphilic microparticle with four discrete hydrophilic patches embedded at the inner corners of a square-shaped hydrophobic outer ring of the particle (4C particle) is fabricated.

Deep Learning-Enhanced Paper-Based Vertical Flow Assay for High-Sensitivity Troponin Detection Using Nanoparticle Amplification.

Successful integration of point-of-care testing (POCT) into clinical settings requires improved assay sensitivity and precision to match laboratory standards. Here, we show how innovations in amplified biosensing, imaging, and data processing, coupled with deep learning, can help improve POCT. To demonstrate the performance of our approach, we present a rapid and cost-effective paper-based high-sensitivity vertical flow assay (hs-VFA) for quantitative measurement of cardiac troponin I (cTnI), a biomarker widely used for measuring acute cardiac damage and assessing cardiovascular risk.

Function-first discovery of high affinity monoclonal antibodies using Nanovial-based plasma B cell screening.

Antibody discovery technologies, essential for research and therapeutic applications, have evolved significantly since the development of hybridoma technology. Various in vitro (display) and in vivo (animal immunization and B cell-sequencing) workflows have led to the discovery of antibodies against diverse antigens. Despite this success, standard display and B-cell sequencing-based technologies are limited to targets that can be produced in a soluble form.

Rapid single-tier serodiagnosis of Lyme disease.

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera.

A Paper-Based Multiplexed Serological Test to Monitor Immunity against SARS-COV-2 Using Machine Learning.

The rapid spread of SARS-CoV-2 caused the COVID-19 pandemic and accelerated vaccine development to prevent the spread of the virus and control the disease. Given the sustained high infectivity and evolution of SARS-CoV-2, there is an ongoing interest in developing COVID-19 serology tests to monitor population-level immunity. To address this critical need, we designed a paper-based multiplexed vertical flow assay (xVFA) using five structural proteins of SARS-CoV-2, detecting IgG and IgM antibodies to monitor changes in COVID-19 immunity levels.

Gelatin methacryloyl granular scaffolds for localized mRNA delivery.

mRNA therapy is the intracellular delivery of messenger RNA (mRNA) to produce desired therapeutic proteins. Developing strategies for local mRNA delivery is still required where direct intra-articular injections are inappropriate for targeting a specific tissue. The mRNA delivery efficiency depends on protecting nucleic acids against nuclease-mediated degradation and safe site-specific intracellular delivery.

Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function.

Validation of a Novel, Rapid Sepsis Diagnostic for Emergency Department Use.

Objectives: To assess the in vitro IntelliSep test, a microfluidic assay that quantifies the state of immune activation by evaluating the biophysical properties of leukocytes, as a rapid diagnostic for sepsis.

Design: Prospective cohort study.

Setting: Five emergency departments (EDs) in Louisiana, Missouri, North Carolina, and Washington.

Passive microfluidic devices for cell separation.

The separation of specific cell populations is instrumental in gaining insights into cellular processes, elucidating disease mechanisms, and advancing applications in tissue engineering, regenerative medicine, diagnostics, and cell therapies. Microfluidic methods for cell separation have propelled the field forward, benefitting from miniaturization, advanced fabrication technologies, a profound understanding of fluid dynamics governing particle separation mechanisms, and a surge in interdisciplinary investigations focused on diverse applications. Cell separation methodologies can be categorized according to their underlying separation mechanisms.

Associating growth factor secretions and transcriptomes of single cells in nanovials using SEC-seq.

Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells.

Fluid dynamics alters liquid-liquid phase separation in confined aqueous two-phase systems.

Liquid-liquid phase separation is key to understanding aqueous two-phase systems (ATPS) arising throughout cell biology, medical science, and the pharmaceutical industry. Controlling the detailed morphology of phase-separating compound droplets leads to new technologies for efficient single-cell analysis, targeted drug delivery, and effective cell scaffolds for wound healing. We present a computational model of liquid-liquid phase separation relevant to recent laboratory experiments with gelatin-polyethylene glycol mixtures.

Injectable Microporous Annealed Crescent-Shaped (MAC) Particle Hydrogel Scaffold for Enhanced Cell Infiltration.

Hydrogels are widely used for tissue engineering applications to support cellular growth, yet the tightly woven structure often restricts cell infiltration and expansion. Consequently, granular hydrogels with microporous architectures have emerged as a new class of biomaterial. Particularly, the development of microporous annealed particle (MAP) hydrogel scaffolds has shown improved stability and integration with host tissue.