Biology subjects

Di Carlo, D.

Publications and source records attributed to Di Carlo, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Hydrogel formulation controls size-dependent accumulation of proteins and nanoparticles in PEG microparticles

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

bioengineering↗

bioRxiv2026

Cell-Cell-Seq resolves contact-associated NK cell activation in defined tumor cell dyads

Cell-cell interactions shape immune recognition, but most single-cell transcriptomic methods measure cells after their interaction history has been lost or inferred. Here we apply Cell-Cell-Seq, a Nanovial-based workflow for sequencing defined cell pairs, to resolve contact-associated activation of natural killer cells paired with leukemia targets. Nanovials enabled controlled dyad formation, incubation, flow enrichment and droplet-based sequencing while reducing uncontrolled partner exchange and aggregation seen in suspension co-culture. Cell-Cell-Seq recovered a reproducible activation program marked by chemokine, cytokine, cytotoxic and immediate-early response genes. Compared with randomly mixed suspension co-culture, defined dyads emphasized contact-proximal activation, whereas suspension co-culture showed stronger features of early overstimulation. Dyad-resolved measurements also benchmarked computational models of cell-cell communication, identifying inferred signalling axes that were recovered and contact-induced programs missed by current approaches. These results establish Cell-Cell-Seq as a scalable strategy for mapping how defined immune-tumour encounters reshape cell state.

bioengineering↗

bioRxiv2026

Systematic mapping of emergent transcriptional states in interacting single-cell dyads by Cell-Cell-seq

Cell-cell interactions drive rapid and heterogeneous changes in gene expression, yet most transcriptomic methods either dissociate cells, losing pair identity and interaction timing, or infer communication indirectly from ligand-receptor co-expression. Here we present Cell-Cell-seq, a scalable workflow for profiling defined cell pairs ("dyads") with single-cell transcriptomic resolution. Cell-Cell-seq uses cavity-containing hydrogel microparticles (Nanovials) to confine two cells, synchronize contact onset, protect fragile conjugates during handling and sorting, and interface directly with droplet-based RNA sequencing. Using antigen-matched prostate tumor cells and engineered T cells as a model system, Cell-Cell-seq captured thousands of tumor-T cell dyads and revealed broad functional and transcriptional heterogeneity across interactions. Dyads unmasked transient activation programs that were obscured in standard well-plate co-culture, consistent with asynchronous contact in bulk assays. To distinguish interaction-induced programs from the composite nature of dyad transcriptomes, we developed a pseudo-mixing framework that generates in silico pseudo-dyads to construct an empirical null distribution under "no interaction," enabling statistically robust identification of emergent genes and partner-resolved attribution of responses. Dyad-resolved analysis further revealed coordinated cross-cell programs, including coupled chemokine expression consistent with bidirectional paracrine signaling and inverse coupling between tumor immunoregulatory programs and T cell activation. Finally, we introduce ccRepair to correct compositional dilution in mixed transcriptomes, improving interpretability while preserving genuine cross-cell coordination. Together, Cell-Cell-seq provides a generalizable platform for dissecting immune synapse biology and mapping interaction-dependent programs across heterogeneous cell populations, with applications in profiling tumor-immune communication and functionally screening immunotherapies.

bioengineering↗

bioRxiv2026

Lab-on-a-3D-Printer for Democratized Reconfigurable Digital Microfluidics

Automation in the life sciences remains dominated by expensive, centralized robotic systems, leaving most laboratories unable to access precision robotic liquid handling. Here we present the Lab-on-a-3D-Printer (Lo3DP), a reconfigurable robotic microfluidic platform that repurposes the motion control, thermal regulation, and open-source programmability of consumer 3D printers to deliver laboratory-grade automation at a hardware cost below $500. By replacing the extruder with a multifunctional magnetic toolhead, Lo3DP actuates ferrofluid droplets with sub-100 {micro}m positioning accuracy and programmable volume control spanning 0.5-25 {micro}L, enabling the full repertoire of digital microfluidic operations within inexpensive laser-cut chips (<$3). We quantitatively validate system performance through long-term droplet transport exceeding 40,000 s without degradation, automated serial dilutions with high linearity (R{superscript 2} [≥] 0.99), and fully automated colorimetric nucleic acid amplification assays whose results match conventional benchtop workflows under tightly regulated isothermal conditions (CV {approx} 0.3%). By harnessing the global economies of scale of consumer 3D printers, Lo3DP provides accurate, reproducible, and programmable assay automation at a fraction of the cost and footprint of conventional systems, establishing a practical path toward desktop-scale, democratized laboratory robotics.

bioengineering↗

bioRxiv2025

A Nanovial-Based Platform for Functional Discovery of Antigen-Reactive TCRs from Unconventional T Cells

Unconventional T cells, such as mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells, recognize non-peptide antigens presented by MR1 and CD1d, respectively, and play pivotal roles in immunity, allowing targeting of cells based on metabolic activity. Although components of the T cell receptors (TCRs) for these unconventional T cells are invariant, significant variability in the CDR3 regions still exist, opening questions as to how TCR sequence and function may be linked, and how to maximize the therapeutic potential of engineered unconventional T cells. Here, we develop a nanovial-based functional screening platform that enables high-throughput discovery of TCRs from unconventional T cells based on direct antigen recognition and cytokine secretion. By selectively labeling nanovials with MR1 and CD1d molecules displaying their cognate ligands, we achieve dose-dependent capture and activation of MAIT and iNKT cells from complex human PBMC samples comprising tens of millions of cells. Using oligonucleotide barcodes conjugated to nanovials encoding the antigen-presenting molecules and loading cytokine capture antibodies, we perform secretion-encoded single-cell sequencing to link TCR identity, gene expression, antigen specificity, and functional response. Applying this method, we isolate rare reactive T cells, recover their TCRs, and validate five novel MAIT TCRs. All five TCRs, when re-expressed in primary T cells, confer antigen-specific cytokine secretion and cytotoxicity. The top two TCRs were evaluated using an in vivo solid tumor model, demonstrating specific tumor homing and efficacy. This function-first strategy offers a powerful tool to uncover functional TCRs from unconventional T cells, yielding a 100% hit rate when secretion-based validation is included as part of the initial screen, unlocking new opportunities for cell-based immunotherapy.

bioengineering↗

bioRxiv2025

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-{gamma} (IFN{gamma}). This system enabled the selective capture, activation, and functional profiling of CAR T cells based on antigen engagement and cytokine secretion. We screened a 32-variant CAR library with diverse intracellular signaling domains, using nanovials to isolate IFN{gamma}-secreting cells after 3- and 12-hour CAR-specific stimulation. IL15RA-containing CARs, particularly IL15RA-CD28, were preferentially enriched in the sorted T cells after 3 hours of stimulation, consistent with early effector activation profiles. By 12 hours, IL15RA-containing constructs remained enriched while other CD40-containing domains showed delayed but substantial enrichment, suggesting prolonged signaling dynamics. The platforms high-throughput capability (>2 million cells screened), compatibility with downstream sequencing, and tunable antigen presentation make it ideal for identifying CAR constructs associated with various time-dependent secretion phenotypes.

bioengineering↗

bioRxiv2025

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. Crucially, these compartments are formed via simple pipetting and centrifugation steps, making the platform highly democratized. We demonstrate the ability of capped nanovials to compartmentalize single mammalian, bacterial, and yeast cells and support growth into colonies, enabling selection based on proliferation and bioproduction. We further show that capped nanovials enhance single-cell secretion assays by reducing molecular crosstalk and increasing signal-to-noise ratios. Importantly, we demonstrate functional co-culture assays by permitting stable confinement of cell pairs, enabling detection and enrichment of antibody-secreting cells based on the ability of their secreted antibodies to activate co-encapsulated reporter T cells, achieving a signal-to-noise ratio of >30 and up to 100% selection purity. By combining the simplicity of standard lab handling with the resolution and throughput of traditional microfluidic compartmentalization approaches, capped nanovials provide a new class of scalable, accessible test tubes for modern single-cell biology.

bioengineering↗

bioRxiv2025

Functionalizing hydrogel nanovials with vesicles mimicking antigen-presenting vesicles and cancer exosomes improves T cell capture and activation

Recent advances have demonstrated the application of microcavity-containing hydrogel microparticles, known as nanovials, for the massively parallel and high-throughput screening of therapeutic T cell populations for adoptive cell therapies. Nanovial cavities coated with peptide-MHC (pMHC) or antigen tetramers selectively bind to their cognate T cell receptor (TCR) or chimeric antigen receptor (CAR) to activate T cells and capture secreted cytokines. However, binding of tetramers or recombinantly expressed antigen by T cells is not always correlated with T cell activation or cytotoxicity as the binding interface is not fully representative of the natural immunological synapse formed between T cells and professional antigen-presenting cells (APCs). Here, we leverage the recent discovery of an ESCRT- and ALIX-binding region (EABR) sequence to generate antigen-presenting vesicles and cancer-mimicking exosomes from standard HEK293T and Expi293F cell cultures. EABR-mediated vesicles present natural, full-length oncologically-relevant membrane proteins embedded in lipid bilayers to functionalize the nanovial cavity with cell-like membranes. These hydrogel nanovials functionalized with the EABR-mediated vesicles show improved T cell capture of 1G4 T cells and enhanced activation of HER2 CAR-T cells compared to hydrogel surfaces functionalized with recombinantly-expressed soluble proteins.

bioengineering↗

bioRxiv2024

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. Earlier efforts address this by localizing capture sites to the nanovial cavity, emulsifying nanovials, or short secretion times to limit secreted antibodies from binding to neighboring nanovials. Here, we demonstrate a method to functionalize nanovials with moieties that impart orthogonal reactivity, enabling conjugation of cell capture antibodies and antigens at different times. We show that by using a strained alkyne moiety to attach cell-capture antibodies via click chemistry to nanovials, we can capture cells and subsequently quantify secretions via biotin-streptavidin linkages. By delaying the loading of antigens onto the nanovials until after cell capture, we were able to ensure high purity (>95%) isolation of hybridoma secreting an antigen-specific antibody in a background of other hybridoma. This approach allows tight temporal control of the secretion measurement, which is independent of the cell loading time, and requires less convective transfer steps. Click chemistry-based coupling further improved cell loading into nanovials by 58% compared to biotin-streptavidin-biotin coupling and caused no reduction in cell viability. We demonstrate an implementation of this system to improve antigen-specific hybridoma screening, yielding an 8-fold improvement in hybridoma enrichment while maintaining a similar workflow complexity. Hybridomas on nanovials sorted into well plates regrew into colonies following sorting using standard fluorescence-activated cell sorting and maintained secretion of antigen-specific antibodies with high purity ([~]90%), as validated via standard enzyme-linked immunosorbent assays. This lab-on-a-particle approach can be applied more generally to decouple cell loading, treatment, or activation, from secretion measurements for single-cell functional assays.

bioengineering↗

bioRxiv2024

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, we fabricated 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). 3D computational fluid dynamics simulations predicted droplet formation dynamics and differing equilibrium conditions depending on the patterning configuration. Experiments recapitulated equilibrium conditions enabling tunable dropicle configurations with reproducible volumes down to [~]200pL templated by the amphiphilic particles. The dropicle configurations depended predominantly on the size of the hydrophilic patches of the 4C particles. This validated modeling approach can inform the design of dropicles with varying volumes and numbers per particle, which can be harnessed in new amplified bioassays for greater sensitivity, dynamic range, and statistical confidence.

bioengineering↗

bioRxiv2024

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. This limitation inhibits the screening of function-inducing antibodies, which require the target to be expressed in cells to monitor function or signaling, and antibodies targeting proteins that maintain their physiological structure only when expressed on cell membranes, such as G-protein coupled receptors (GPCRs). A high-throughput two-cell screening workflow, which localizes an antibody-secreting cell (ASC) and a cell expressing the target protein in a microenvironment, can overcome these challenges. To make function-first plasma cell-based antibody discovery accessible and scalable, we developed hydrogel Nanovials that can capture single plasma cells, target-expressing cells, and plasma cell secretions (antibodies). The detection and isolation of Nanovials harboring the antigen-specific plasma cells are then carried out using a flow cytometry cell sorter - an instrument that is available in most academic centers and biopharmaceutical companies. The antibody discovery workflow employing Nanovials was first validated in the context of two different cell membrane-associated antigens produced in recombinant form. We analyzed over 40,000 plasma cells over two campaigns and were able to identify a diversity of binders that i) exhibited high affinity (picomolar) binding, ii) targeted multiple non-overlapping epitopes and iii) demonstrated high developability scores. A campaign using the two-cell assay targeting the immune checkpoint membrane protein PD-1 yielded cell binders with similar EC50s to clinically used Pembrolizumab and Nivolumab. The highest selectivity for binders was observed for sorted events corresponding with the highest signal bound to target cells on Nanovials. Overall, Nanovials can provide a strong foundation for function-first antibody discovery, yielding direct cell binding information and quantitative data on prioritization of hits with flexibility for additional functional readouts in the future.

bioengineering↗

bioRxiv2024

Linking single-cell transcriptomes with secretion using secretion-encoded single-cell sequencing (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. We provide a protocol that enables linking the Secretion amount of a target protein EnCoded (SEC) by thousands of single cells with transcriptional sequencing (seq). SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials, and flow cytometry and single-cell RNA-sequencing platforms for readout. Cells on Nanovials can be sorted based on viability, secretion amount, or other surface markers without fixation or permeabilization, and cell and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T-cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells, and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes under 4 hours, and once the desired incubation time is over, staining, cell sorting, and emulsion generation for scRNA-seq can also be completed in under 4 hours. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated, and how it can be engineered to make better therapies.

bioengineering↗

bioRxiv2024

Super homotypic targeting by exosome surface engineering

Homotypic targeting is the inherent ability of cells for preferential interaction with cells of similar or identical types, a phenomenon commonly seen in cell adhesion, tissue formation, and immune responses. Unfortunately, its full potential remains largely untapped. Here we introduce an approach to drastically boost the homotypic targeting capabilities of cells via exosomes (nanoscale extracellular vesicles secreted by cells). By engineering exosome surfaces with lanthanides, we amplify specific cell-exosome interactions by more than 25-fold, significantly accelerating the selective capture of exosomes by cells of the same lineage. This substantial enhancement in cellular homophilicity opens up an entirely new class of applications, two of which we showcase here with unprecedented performance: using cells to detect specific exosomes and using exosomes to detect specific cells. The concept of "super homotypic targeting" offers enormous potential to transform cancer diagnostics, immunotherapy, targeted drug delivery, tissue engineering, and vaccine development.

cell biology↗

bioRxiv2024

Flow zoometry of Drosophila

Drosophila serves as a highly valuable model organism across numerous fields including genetics, immunology, neuroscience, cancer biology, and developmental biology. Central to Drosophila-based biological research is the ability to perform comprehensive genetic or chemical screens. However, this research is often limited by its dependence on laborious manual handling and analysis, making it prone to human error and difficult to discern statistically significant or rare events amid the noise of individual variations resulting from genetic and environmental factors. In this article we present flow zoometry, a whole-animal equivalent of flow cytometry for large-scale, individual-level, high-content screening of Drosophila. Our flow zoometer automatically clears the tissues of Drosophila melanogaster, captures three-dimensional (3D) multi-color fluorescence tomograms of single flies with single-cell volumetric resolution at an unprecedented throughput of over 1,000 animals within 48 hours (24 hr for clearing; 24 hr for imaging), and performs AI-enhanced data-driven analysis - a task that would traditionally take months or years with manual techniques. To demonstrate its broad applications, we employed the flow zoometer in various laborious screening assays, including those in toxicology, genotyping, and tumor screening. Flow zoometry represents a pivotal evolution in high-throughput screening technology: previously from molecules to cells, now from cells to whole animals. This advancement serves as a foundational platform for "statistical spatial biology", to improve empirical precision and enable serendipitous discoveries across various fields of biology.

bioengineering↗

bioRxiv2023

Single-tier point-of-care serodiagnosis of Lyme disease

Point-of-care (POC) serological testing provides actionable information for several difficult to diagnose illnesses, empowering distributed health systems. Accessible and adaptable diagnostic platforms that can assay the repertoire of antibodies formed against pathogens are essential to drive early detection and improve patient outcomes. Here, we report a POC serologic test for Lyme disease (LD), leveraging synthetic peptides tuned to be highly specific to the LD antibody repertoire across patients and compatible with a paper-based platform for rapid, reliable, and cost-effective diagnosis. A subset of antigenic epitopes conserved across Borrelia burgdorferi genospecies and targeted by IgG and IgM antibodies, were selected based on their seroreactivity to develop a multiplexed panel for a single-step measurement of combined IgM and IgG antibodies from LD patient sera. Multiple peptide epitopes, when combined synergistically using a machine learning-based diagnostic model, yielded a high sensitivity without any loss in specificity. We blindly tested the platform with samples from the U.S. Centers for Disease Control & Prevention (CDC) LD repository and achieved a sensitivity and specificity matching the lab-based two-tier results with a single POC test, correctly discriminating cross-reactive look-alike diseases. This computational LD diagnostic test can potentially replace the cumbersome two-tier testing paradigm, improving diagnosis and enabling earlier effective treatment of LD patients while also facilitating immune monitoring and surveillance of the disease in the community.

bioengineering↗

bioRxiv2023

Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. We developed a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach was applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibited distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintained high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improved heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

bioengineering↗

bioRxiv2023

Amphiphilic particle-stabilized nanoliter droplet reactors with a multi-modal portable reader for distributive biomarker quantification

Compartmentalization, leveraging microfluidics, enables highly sensitive assays; but the requirement for significant infrastructure for their design, build, and operation limits access. Newer multi-material particle-based technologies thermodynamically stabilize monodisperse droplets as individual reaction compartments with simple liquid handling steps, precluding the need for expensive microfluidic equipment. Here, we further improve the accessibility of this lab on a particle technology to resource-limited settings by combining this assay system with a portable multi-modal reader, thus enabling nanoliter droplet assays in an accessible platform. We show the utility of this platform in measuring N-terminal propeptide B-type natriuretic peptide (NT-proBNP), a heart failure biomarker, in complex medium and patient samples. We report a limit of detection of [~]0.05 ng/ml and a linear response between 0.2 - 2 ng/ml in spiked plasma samples. We also show that, owing to the plurality of measurements per sample, "swarm" sensing acquires better statistical quantitation with a portable reader. Monte Carlo simulations show the increasing capability of this platform to differentiate between negative and positive samples, i.e. below or above the clinical cut-off for acute heart failure ([~]0.1ng/ml), as a function of the number of particles measured. Our platform measurements correlate with gold standard ELISA measurement in cardiac patient samples, and achieve lower variation in measurement across samples compared to the standard well plate-based ELISA. Thus, we show the capabilities of a cost-effective droplet-reader system in accurately measuring biomarkers in nanoliter droplets for diseases that disproportionally affect underserved communities in resource-limited settings.

bioengineering↗

bioRxiv2023

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

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired {beta}-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques. One-sentence SummaryAffinity and secretion-based screening of antigen-specific T cells using nanovials defines a functional TCR repertoire

bioengineering↗