Chan Zuckerberg Biohub Chicago

Continuous measurement of proteins in vivo is important for real-time disease management and prevention. Implantable sensors for monitoring small molecules such as glucose have been available for more than a decade. However, analysis of proteins remains an unmet need because the lower physiological levels require that sensors have high affinities, which are linked to long complexation half-lives ( t 1/2 ~20 hours) and slow equilibration when concentrations decrease. We report active-reset sensors by use of high-frequency oscillations to accelerate dissociation, which enables regeneration of the unbound form of the sensor within 1 minute. When implemented within implanted devices, these sensors allow for real-time, in vivo monitoring of proteins within interstitial fluid. Active-reset protein sensors track biomarker levels on a physiological timescale for inflammation monitoring in living animals.

Rapid and accurate detection of DNA from disease-causing pathogens is essential for controlling the spread of infections and administering timely treatments. While traditional molecular diagnostics techniques like PCR are highly sensitive, they include nucleic acid amplification and many need to be performed in centralized laboratories, limiting their utility in point-of-care settings. Recent advances in CRISPR-based diagnostics (CRISPR-Dx) have demonstrated the potential for highly specific molecular detection, but the sensitivity is often constrained by the slow trans-cleavage activity of Cas enzymes, necessitating preamplification of target nucleic acids. In this study, we present a CRISPR-Cascade assay that overcomes these limitations by integrating a positive feedback loop that enables nucleic acid amplification-free detection of pathogenic DNA at atto-molar levels and achieves a signal-to-noise ratio greater than 1.3 within just 10 min. The versatility of the assay is demonstrated through the detection of bloodstream infection pathogens, including Methicillin-Sensitive Staphylococcus aureus (MSSA), Methicillin-Resistant Staphylococcus aureus (MRSA), Escherichia coli , and Hepatitis B Virus (HBV) spiked in whole blood samples. Additionally, we introduce a multiplexing OR-function logic gate, further enhancing the potential of the CRISPR-Cascade assay for rapid and accurate diagnostics in clinical settings. Our findings highlight the ability of the CRISPR-Cascade assay to provide highly sensitive and specific molecular detection, paving the way for advanced applications in point-of-care diagnostics and beyond.

Stem cells hold promise in regenerative medicine due to their ability to proliferate and differentiate into various cell types. However, their self-renewal and multipotency also raise concerns about their tumorigenicity during and post-therapy. Indeed, multiple studies have reported the presence of stem cell-derived tumors in animal models and clinical administrations. Therefore, the assessment of tumorigenicity is crucial in evaluating the safety of stem cell-derived therapeutic products. Ideally, the assessment needs to be performed rapidly, sensitively, cost-effectively, and scalable. This article reviews various approaches for assessing tumorigenicity, including animal models, soft agar culture, PCR, flow cytometry, and microfluidics. Each method has its advantages and limitations. The selection of the assay depends on the specific needs of the study and the stage of development of the stem cell-derived therapeutic product. Combining multiple assays may provide a more comprehensive evaluation of tumorigenicity. Future developments should focus on the optimization and standardization of microfluidics-based methods, as well as the integration of multiple assays into a single platform for efficient and comprehensive evaluation of tumorigenicity.

century, it is only in recent years where such unique classes of materials have begun to find use in bioelectronics-itself a burgeoning area of research. Inspired by the natural ability of biological tissue to self-repair, self-healing materials play a multifaceted role in the context of soft, wireless bioelectronic systems, in that they can not only serve as a protective outer shell or substrate for the internal electronic circuitry-analogous to the mechanical barrier that skin provides for the human body-but also, and most importantly, act as an active sensing safeguard against mechanical damage to preserve device functionality and enhance overall durability. This perspective presents the historical overview, general design principles, recent developments, and future outlook of self-healing materials for bioelectronic devices, which integrates topics in many research disciplines-from materials science and chemistry to electronics and bioengineering-together.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Reagent-free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid-based reagent-free electronic sensing system. The signal transduction is based on the kinetics of an electrode-tethered molecular pendulum-a rigid double stranded DNA with one of the strands displaying an analyte-binding aptamer and the other featuring a redox probe-that exhibits field-induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross-reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.

Significant advances in science and engineering often emerge at the intersections of disciplines. Nanoscience and nanotechnology are inherently interdisciplinary, uniting researchers from chemistry, physics, biology, medicine, materials science, and engineering. This convergence has fostered novel ways of thinking and enabled the development of materials, tools, and technologies that have transformed both basic and applied research, as well as how we address critical societal challenges. In this Nano Focus, we pose and explore 33 questions whose answers could profoundly impact fields such as energy, electronics, the environment, optics, and medicine. These questions highlight the need for deeper foundational understanding, improved tools and techniques, and innovative applications─each with significant societal relevance. Together, they represent a global call-to-action for the scientific community.

Abstract Immune Cellular Therapies (ICT) have revolutionized the treatment of blood cancer and are beginning to show positive outcomes in treating solid tumors. Despite these successes, ICT faces significant challenges, including tumor accessibility, lengthy manufacturing turnaround, and limited long‐term effectiveness. Recent advancements in nanomaterials, particularly nanoparticles, have offered promising solutions to these issues. This perspective introduces the current ICT manufacturing pipeline with a focus on solid tumors and showcases recent nanomaterial‐mediated practices to enhance ICT. These efforts include the use of cell‐targeting magnetic nanoparticles for non‐invasive target identification, lipid nanoparticles for in vivo immune cell stimulation, as well as nanoparticle‐mediated gene editing and cytokine delivery to enhance immune cell fitness. By better integrating nanoparticles into the design and manufacturing pipelines, we envision that the next generation of ICT could be faster, more efficient, and capable of targeting a broad spectrum of cancers and inflammatory diseases.

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalization─installation of a covalent warhead─with mRNA display and showcases its application in targeted covalent ligand discovery.

Nanoneedles are a useful tool for delivering exogenous biomolecules to cells. Although therapeutic applications have been explored, the mechanism regarding how cells interact with nanoneedles remains poorly studied. Here, we present a new approach for the generation of nanoneedles, validated their usefulness in cargo delivery, and studied the underlying genetic modulators during delivery. We fabricated arrays of nanoneedles based on electrodeposition and quantified its efficacy of delivery using fluorescently labeled proteins and siRNAs. Notably, we revealed that our nanoneedles caused the disruption of cell membranes, enhanced the expression of cell–cell junction proteins, and downregulated the expression of transcriptional factors of NFκB pathways. This perturbation trapped most of the cells in G2 phase, in which the cells have the highest endocytosis activities. Taken together, this system provides a new model for the study of interactions between cells and high-aspect-ratio materials.

Single-cell RNA sequencing (scRNA-seq) datasets contain true single cells, or singlets, in addition to cells that coalesce during the protocol, or doublets. Identifying singlets with high fidelity in scRNA-seq is necessary to avoid false negative and false positive discoveries. Although several methodologies have been proposed, they are typically tested on highly heterogeneous datasets and lack a priori knowledge of true singlets. Here, we leveraged datasets with synthetically introduced DNA barcodes for a hitherto unexplored application: to extract ground-truth singlets. We demonstrated the feasibility of our framework, "singletCode," to evaluate existing doublet detection methods across a range of contexts. We also leveraged our ground-truth singlets to train a proof-of-concept machine learning classifier, which outperformed other doublet detection algorithms. Our integrative framework can identify ground-truth singlets and enable robust doublet detection in non-barcoded datasets.

Structural nanomedicines are engineered constructs that arrange therapeutic components into well-defined architectures to maximize efficacy. Their multivalent, multifunctional design offers key advantages over unstructured formulations, including targeted delivery, expanded therapeutic windows, and enhanced target engagement. The mRNA COVID-19 vaccines exemplify their transformative potential. However, structural precision varies, and more well-defined architectures will streamline optimization, manufacturing, and regulation. Unlike small molecule drugs, nanomedicines within a batch are not identical. Identifying the most effective, least toxic structures will advance our understanding of structure-function relationships and therapeutic mechanisms. This work highlights structural nanomedicines─small molecules, nucleic acids, and biologics─to galvanize the field and drive innovation toward even safer, more effective treatments that benefit patients.

While existing synthetic technologies for ex vivo T-cell activation face challenges like suboptimal expansion rates and low effectiveness, artificial antigen-presenting cells (aAPCs) hold great promise for enhanced T-cell based therapies. In particular, gold nanoparticles (AuNPs), known for their biocompatibility, ease of synthesis, and versatile surface chemistry, are strong candidates for use as nanoscale aAPCs. In this study, we developed spiky AuNPs with branched geometries to present activating ligands to primary human T-cells. The special structure of spiky AuNPs enhances biomolecule loading capacity and significantly improves T-cell activation through multivalent binding of costimulatory ligands and receptors. Our spiky AuNPs outperform existing systems including Dynabeads and soluble activators by promoting greater polyclonal expansion of T-cells, boosting sustained cytokine production, and generating highly functional T-cells with reduced exhaustion. In addition, spiky AuNPs effectively activate and expand CD19 CAR-T cells while demonstrating increased in vitro cytotoxicity against target cells using fewer effector cells than Dynabeads. This study underscores the potential of spiky AuNPs as a powerful tool, bringing new opportunities to adoptive cell therapy applications.

Intracellular electrophysiology is essential in neuroscience, cardiology, and pharmacology for studying cells' electrical properties. Traditional methods like patch-clamp are precise but low-throughput and invasive. Nanoelectrode Arrays (NEAs) offer a promising alternative by enabling simultaneous intracellular and extracellular action potential (iAP and eAP) recordings with high throughput. However, accessing intracellular potentials with NEAs remains challenging. This study presents an AI-supported technique that leverages thousands of synchronous eAP and iAP pairs from stem-cell-derived cardiomyocytes on NEAs. Our analysis revealed strong correlations between specific eAP and iAP features, such as amplitude and spiking velocity, indicating that extracellular signals could be reliable indicators of intracellular activity. We developed a physics-informed deep learning model to reconstruct iAP waveforms from extracellular recordings recorded from NEAs and Microelectrode arrays (MEAs), demonstrating its potential for non-invasive, long-term, high-throughput drug cardiotoxicity assessments. This AI-based model paves the way for future electrophysiology research across various cell types and drug interactions.

Continuous biomarker monitoring is revolutionizing chronic disease management, with glucose monitoring for diabetes as the primary example. Given the success of this approach, a transition to continuous protein monitoring (CPM, a real-time, implantable or wearable technology) could similarly advance precision medicine. In this work, we review state-of-the-art CPM platforms and their prospective clinical impact across both chronic disorders-metabolic, cardiovascular, autoimmune, and neurodegenerative-and acute crises, such as sepsis and transplant dysfunction. We also highlight remaining barriers to widespread adoption, including sensor stability, robust machine learning models for live interpretation, and responsible data handling for patient privacy. With continued engineering and clinical validation, emerging biosensor technologies could transform disease management, facilitating earlier interventions and individualizing treatment strategies, ultimately improving patient outcomes.

Abstract Reagent‐free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid‐based reagent‐free electronic sensing system. The signal transduction is based on the kinetics of an electrode‐tethered molecular pendulum—a rigid double stranded DNA with one of the strands displaying an analyte‐binding aptamer and the other featuring a redox probe—that exhibits field‐induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross‐reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.

Nanomaterials have extensive applications in the development of sensitive biosensors, but the influence of their specific structural properties remains unclear. This work presents a platform that can provide mechanistic insight into how nanostructured electrodes improve the performance of electrochemical biosensors. We designed nanoelectrodes with sub-10 nm spike features through a combination of top-down lithography and solution-based synthesis. These anisotropic structures facilitated rapid electron-transfer, minimized biofouling, and promoted efficient target capture. Using these spiky nanoelectrodes in a biosensor, we detected bacterial mRNA at aM-levels and within 3 min. Our findings reveal the mechanism underlying signal enhancement from high-curvature regions on nanostructured electrodes, highlighting the structure-property relationships of nanostructures in electrochemical sensing.

Neuronal control of skeletal muscle function is ubiquitous across species for locomotion and doing work. In particular, emergent behaviors of neurons in biohybrid neuromuscular systems can advance bioinspired locomotion research. Although recent studies have demonstrated that chemical or optogenetic stimulation of neurons can control muscular actuation through the neuromuscular junction (NMJ), the correlation between neuronal activities and resulting modulation in the muscle responses is less understood, hindering the engineering of high-level functional biohybrid systems. Here, we developed NMJ-based biohybrid crawling robots with optogenetic mouse motor neurons, skeletal muscles, 3D-printed hydrogel scaffolds, and integrated onboard wireless micro-light-emitting diode (μLED)-based optoelectronics. We investigated the coupling of the light stimulation and neuromuscular actuation through power spectral density (PSD) analysis. We verified the modulation of the mechanical functionality of the robot depending on the frequency of the optical stimulation to the neural tissue. We demonstrated continued muscle contraction up to 20 minutes after a 1-minute-long pulsed 2-hertz optical stimulation of the neural tissue. Furthermore, the robots were shown to maintain their mechanical functionality for more than 2 weeks. This study provides insights into reliable neuronal control with optoelectronics, supporting advancements in neuronal modulation, biohybrid intelligence, and automation.

Numerous human cancers have exhibited the ability to elude immune checkpoint blockade (ICB) therapies. This type of resistance can be mediated by immune-suppressive macrophages that limit antitumor immunity in the tumor microenvironment (TME). Here, we elucidate a strategy to shift macrophages into a proinflammatory state that down-regulates V domain immunoglobulin suppressor of T cell activation (VISTA) via inhibiting AhR and IRAK1. We used a high-throughput microfluidic platform combined with a genome-wide CRISPR knockout screen to identify regulators of VISTA levels. Functional characterization showed that the knockdown of these hits diminished VISTA surface levels on macrophages and sustained an antitumor phenotype. Furthermore, targeting of both AhR and IRAK1 in mouse models overcame resistance to ICB treatment. Tumor immunophenotyping indicated that infiltration of cytotoxic CD8 + cells, natural killer cells, and antitumor macrophages was substantially increased in treated mice. Collectively, AhR and IRAK1 are implicated as regulators of VISTA that coordinate a multifaceted barrier to antitumor immune responses.

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of the Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that the RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics are controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.