Phenotypic targeting using magnetic nanoparticles for rapid characterization of cellular proliferation regulators.

Genome-wide CRISPR screens have provided a systematic way to identify essential genetic regulators of a phenotype of interest with single-cell resolution. However, most screens use live/dead readout of viability to identify factors of interest. Here, we describe an approach that converts cell proliferation into the degree of magnetization, enabling downstream microfluidic magnetic sorting to be performed. We performed a head-to-head comparison and verified that the magnetic workflow can identify the same hits from a traditional screen while reducing the screening period from 4 weeks to 1 week. Taking advantage of parallelization and performance, we screened multiple mesenchymal cancer cell lines for their dependency on cell proliferation. We found and validated pan- and cell-specific potential therapeutic targets. The method presented provides a nanoparticle-enabled approach means to increase the breadth of data collected in CRISPR screens, enabling the rapid discovery of drug targets for treatment.

Identification of druggable regulators of cell secretion via a kinome-wide screen and high-throughput immunomagnetic cell sorting.

The identification of genetic regulators of cell secretions is challenging because it requires the sorting of a large number of cells according to their secretion patterns. Here we report the development and applicability of a high-throughput microfluidic method for the analysis of the secretion levels of large populations of immune cells. The method is linked with a kinome-wide loss-of-function CRISPR screen, immunomagnetically sorting the cells according to their secretion levels, and the sequencing of their genomes to identify key genetic modifiers of cell secretion. We used the method, which we validated against flow cytometry for cytokines secreted from primary mouse CD4+ (cluster of differentiation 4-positive) T cells, to discover a subgroup of highly co-expressed kinase-coding genes that regulate interferon-gamma secretion by these cells. We validated the function of the kinases identified using RNA interference, CRISPR knockouts and kinase inhibitors and confirmed the druggability of selected kinases via the administration of a kinase inhibitor in an animal model of colitis. The technique may facilitate the discovery of regulatory mechanisms for immune-cell activation and of therapeutic targets for autoimmune diseases.

CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality.

A major impediment to the characterization of mtDNA repair mechanisms in comparison to nuclear DNA repair mechanisms is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of an mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox), we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted doxorubicin (Dox) screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentiality of our top hit, WRNIP1─observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.

Apta FastZ: An Algorithm for the Rapid Identification of Aptamers with Defined Binding Affinities.

Real-time biomolecular monitoring requires biosensors based on affinity reagents, such as aptamers, with moderate to low affinities for the best binding dynamics and signal gain. We recently reported Pro-SELEX, an approach that utilizes parallelized SELEX and high-content bioinformatics for the selection of aptamers with predefined binding affinities. The Pro-SELEX pipeline relies on an algorithm, termed AptaZ, that can predict the binding affinities of selected aptamers. The original AptaZ algorithm is computationally complex and slows the overall throughput of Pro-SELEX. Here, we present Apta FastZ, a rapid equivalent of AptaZ. The Apta FastZ algorithm considers the spare nature of the sequences from SELEX and is coded to avoid unnecessary comparison between sequences. As a result, Apta FastZ achieved a 10 to 40-fold faster computing speed compared to the original AptaZ algorithm while maintaining identical outcomes, allowing the bioinformatics to be completed within 1-10 h for large-scale data sets. We further validated the affinity of myeloperoxidase aptamers predicted by Apta FastZ by experiments and observed a high level of linear correlation between predicted scores and measured affinities. Taken together, the implementation of Apta FastZ could greatly accelerate the current Pro-SELEX workflow, allowing customized aptamers to be discovered within 3 days using preselected DNA libraries.

Self-Assembled Homopolymeric Spherulites from Small Molecules in Solution.

Polymeric spherulites are typically formed by melt crystallization: spherulitic growth in solution is rare and requires complex polymers and dilute solutions. Here, we report the mild and unique formation of luminescent spherulites at room temperature via the simple molecule benzene-1,4-dithiol (BDT). Specifically, BDT polymerized into oligomers (PBDT) via disulfide bonds and assembled into uniform supramolecular nanoparticles in aqueous buffer; these nanoparticles were then dissolved back into PBDT in a good solvent (i.e., dimethylformamide) and underwent chain elongation to form spherulites (rPBDT) in 10 min. The spherulite geometry was modulated by changing the PBDT concentration and reaction time. Due to the step-growth polymerization and reorganization of PBDT, these spherulites not only exhibited robust structure but also showed broad clusterization-triggered emission. The biocompatibility and efficient cellular uptake of the spherulites further underscore their value as traceable drug carriers. This system provides a new pathway for designing versatile superstructures with value for hierarchical assembly of small molecules into a complicated biological system.

CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality.

A major impediment to the characterization of mtDNA repair mechanisms, in comparison to nuclear DNA repair mechanisms, is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of a mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox) we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted Dox screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentially of our top hit - WRNIP1 - observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.

A magneto-activated nanoscale cytometry platform for molecular profiling of small extracellular vesicles.

Exosomal PD-L1 (exoPD-L1) has recently received significant attention as a biomarker predicting immunotherapeutic responses involving the PD1/PD-L1 pathway. However, current technologies for exosomal analysis rely primarily on bulk measurements that do not consider the heterogeneity found within exosomal subpopulations. Here, we present a nanoscale cytometry platform NanoEPIC, enabling phenotypic sorting and exoPD-L1 profiling from blood plasma. We highlight the efficacy of NanoEPIC in monitoring anti-PD-1 immunotherapy through the interrogation of exoPD-L1. NanoEPIC generates signature exoPD-L1 patterns in responders and non-responders. In mice treated with PD1-targeted immunotherapy, exoPD-L1 is correlated with tumor growth, PD-L1 burden in tumors, and the immune suppression of CD8+ tumor-infiltrating lymphocytes. Small extracellular vesicles (sEVs) with different PD-L1 expression levels display distinctive inhibitory effects on CD8 + T cells. NanoEPIC offers robust, high-throughput profiling of exosomal markers, enabling sEV subpopulation analysis. This platform holds the potential for enhanced cancer screening, personalized treatment, and therapeutic response monitoring.

A high-dimensional microfluidic approach for selection of aptamers with programmable binding affinities.

Aptamers are being applied as affinity reagents in analytical applications owing to their high stability, compact size and amenability to chemical modification. Generating aptamers with different binding affinities is desirable, but systematic evolution of ligands by exponential enrichment (SELEX), the standard for aptamer generation, is unable to quantitatively produce aptamers with desired binding affinities and requires multiple rounds of selection to eliminate false-positive hits. Here we introduce Pro-SELEX, an approach for the rapid discovery of aptamers with precisely defined binding affinities that combines efficient particle display, high-performance microfluidic sorting and high-content bioinformatics. Using the Pro-SELEX workflow, we were able to investigate the binding performance of individual aptamer candidates under different selective pressures in a single round of selection. Using human myeloperoxidase as a target, we demonstrate that aptamers with dissociation constants spanning a 20-fold range of affinities can be identified within one round of Pro-SELEX.

Biomolecular sensors for advanced physiological monitoring.

Body-based biomolecular sensing systems, including wearable, implantable and consumable sensors allow comprehensive health-related monitoring. Glucose sensors have long dominated wearable bioanalysis applications owing to their robust continuous detection of glucose, which has not yet been achieved for other biomarkers. However, access to diverse biological fluids and the development of reagentless sensing approaches may enable the design of body-based sensing systems for various analytes. Importantly, enhancing the selectivity and sensitivity of biomolecular sensors is essential for biomarker detection in complex physiological conditions. In this Review, we discuss approaches for the signal amplification of biomolecular sensors, including techniques to overcome Debye and mass transport limitations, and selectivity improvement, such as the integration of artificial affinity recognition elements. We highlight reagentless sensing approaches that can enable sequential real-time measurements, for example, the implementation of thin-film transistors in wearable devices. In addition to sensor construction, careful consideration of physical, psychological and security concerns related to body-based sensor integration is required to ensure that the transition from the laboratory to the human body is as seamless as possible.

Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting.

The clinical use of tumour-infiltrating lymphocytes for the treatment of solid tumours is hindered by the need to obtain large and fresh tumour fractions, which is often not feasible in patients with unresectable tumours or recurrent metastases. Here we show that circulating tumour-reactive lymphocytes (cTRLs) can be isolated from peripheral blood at high yield and purity via microfluidic immunomagnetic cell sorting, allowing for comprehensive downstream analyses of these rare cells. We observed that CD103 is strongly expressed by the isolated cTRLs, and that in mice with subcutaneous tumours, tumour-infiltrating lymphocytes isolated from the tumours and rapidly expanded CD8+CD103+ cTRLs isolated from blood are comparably potent and respond similarly to immune checkpoint blockade. We also show that CD8+CD103+ cTRLs isolated from the peripheral blood of patients and co-cultured with tumour cells dissociated from their resected tumours resulted in the enrichment of interferon-γ-secreting cell populations with T-cell-receptor clonotypes substantially overlapping those of the patients' tumour-infiltrating lymphocytes. Therapeutically potent cTRLs isolated from peripheral blood may advance the clinical development of adoptive cell therapies.

Efficient Delivery of Biological Cargos into Primary Cells by Electrodeposited Nanoneedles via Cell-Cycle-Dependent Endocytosis.

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.

Monitoring Cardiac Biomarkers with Aptamer-Based Molecular Pendulum Sensors.

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.

Digital microfluidics as an emerging tool for bacterial protocols.

Bacteria are widely studied in various research areas, including synthetic biology, sequencing and diagnostic testing. Protocols involving bacteria are often multistep, cumbersome and require access to a long list of instruments to perform experiments. In order to streamline these processes, the fluid handling technique digital microfluidics (DMF) has provided a miniaturized platform to perform various steps of bacterial protocols from sample preparation to analysis. DMF devices can be paired/interfaced with instrumentation such as microscopes, plate readers, and incubators, demonstrating their versatility with existing research tools. Alternatively, DMF instruments can be integrated into all-in-one packages with on-chip magnetic separation for sample preparation, heating/cooling modules to perform assay steps and cameras for absorbance and/or fluorescence measurements. This perspective outlines the beneficial features DMF offers to bacterial protocols, highlights limitations of current work and proposes future directions for this tool's expansion in the field.

Ultrathroughput immunomagnetic cell sorting platform.

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.

Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis.

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2-a gene encoding a secreted protein acting as a cellular migration cue-as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype, resulting in enhanced cancer progression in xenograft models.

Capillary-Assisted Molecular Pendulum Bioanalysis.

The development of robust biosensing strategies that can be easily implemented in everyday life remains a challenge for the future of modern biosensor research. While several reagentless approaches have attempted to address this challenge, they often achieve user-friendliness through sacrificing sensitivity or universality. While acceptable for certain applications, these trade-offs hinder the widespread adoption of reagentless biosensing technologies. Here, we report a novel approach to reagentless biosensing that achieves high sensitivity, rapid detection, and universality using the SARS-CoV-2 virus as a model target. Universality is achieved by using nanoscale molecular pendulums, which enables reagentless electrochemical biosensing through a variable antibody recognition element. Enhanced sensitivity and rapid detection are accomplished by incorporating the coffee-ring phenomenon into the sensing scheme, allowing for target preconcentration on a ring-shaped electrode. Using this approach, we obtained limits of detection of 1 fg/mL and 20 copies/mL for the SARS-CoV-2 nucleoproteins and viral particles, respectively. In addition, clinical sample analysis showed excellent agreement with Ct values from PCR-positive SARS-CoV-2 patients.

Nanoparticle Amplification Labeling for High-Performance Magnetic Cell Sorting.

Magnetic cell sorting is an enabling tool for the isolation of specific cellular subpopulations for downstream applications and requires the cells to be labeled by a sufficient number of magnetic nanoparticles to leverage magnetophoresis for efficient separation. This requirement makes it challenging to target weakly expressed biomarkers. Here, we developed a new approach that selectively and efficiently amplifies the magnetic labeling on cells through sequentially connected antibodies and nanoparticles delivered to the surface or interior of the cell. Using this approach, we achieved amplification up to 100-fold for surface and intracellular markers. We also demonstrated the utility of this assay for enabling high-performance magnetic cell sorting when it is applied to the analysis of rare tumor cells for cancer diagnosis and the purification of transfected CAR T cells for immunotherapy. The data presented demonstrate a useful tool for the stratification of rare cell subpopulations.

PillarX: A Microfluidic Device to Profile Circulating Tumor Cell Clusters Based on Geometry, Deformability, and Epithelial State.

Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The PillarX device described here is a bimodular microfluidic device (Pillar-device and an X-magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar-device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X-device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell-cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.