Mechanical regulation of extracellular vesicle activity during tumour progression.

Extracellular vesicles (EVs) are naturally occurring membrane-bound vesicles secreted by cells. Functionalized with surface-targeting molecules and carrying signalling proteins and nucleic acids as cargo, EVs can rewire pathways and alter biological processes in recipient cells. Tumour-derived EVs have key roles in cancer progression, particularly in metastasis, by promoting tumour cell invasion and the establishment of pre-metastatic niches.

Extracellular Vesicles for Clinical Diagnostics: From Bulk Measurements to Single-Vesicle Analysis.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, signaling pathways, and disease pathogenesis by transporting biomolecules such as DNA, RNA, proteins, and lipids derived from their cells of origin, and they have demonstrated substantial potential in clinical applications. Their clinical significance underscores the need for sensitive methods to fully harness their diagnostic potential. In this comprehensive review, we explore EV heterogeneity related to biogenesis, structure, content, origin, sample type, and function roles; the use of EVs as disease biomarkers; and the evolving landscape of EV measurement for clinical diagnostics, highlighting the progression from bulk measurement to single vesicle analysis.

Brain biomarker profiles vary with semi-synthetic and grain-based diets in healthy and mTBI mice.

Across a range of neurological disorders, there is a growing appreciation for how the gut influences brain health, but few ways of monitoring its effects. Although nutrition influences traumatic brain injury (TBI) recovery, its influence on biomarkers-whether as an intervention or confounder-is poorly understood. Beyond specialized diets, standard rodent diets may also affect brain function.

Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries.

Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds.

Combining time domain modulation optofluidics and high dynamic range imaging for multiplexed, high throughput digital droplet assays.

Digital enzyme-linked immunoassays (dELISA) have been successfully applied to the ultrasensitive quantification of analytes, including nucleic acids, proteins, cells, and extracellular vesicles, achieving robust detection limits in complex clinical specimens such as blood, and demonstrating utility across a broad range of clinical applications. The ultrasensitivity of dELISA comes from partitioning single analytes, captured onto a microbead, into millions of compartments so that they can be counted individually. There is particular interest in using dELISA for multiplexed measurements, but generating and detecting the billions of compartments necessary to perform multiplexed ultrasensitive dELISA remains a challenge.

Agarose Microgel-Based In Situ Cleavable Immuno-Rolling Circle Amplification for Multiplexed Single-Molecule Quantitation on Single Extracellular Vesicles.

We have developed a platform for the multiplexed and ultrasensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call GDEVA─Agarose microel-based igital single-molecule-single ssay. GDEVA achieves single-molecule sensitivity and moderate multiplexing (demonstrated 3-plex), and can achieve a throughput of ∼10 EVs per minute necessary to resolve EVs directly in human plasma when read out using flow cytometry. Our platform integrates a rolling circle amplification (RCA) immunoassay of EV surface proteins, which are cleaved from single EVs, and amplified within agarose microgels, followed by flow cytometry-based readout or imaging after fluorescence-activated cell sorting (FACS).

Enhanced Accumulation and Penetration of Magnetic Nanoclusters in Tumors Using an 8-Magnet Halbach Array Leads to Improved Cancer Treatment.

Nanoparticles have gained attention as drug delivery vehicles for cancer treatment, but often struggle with poor tumor accumulation and penetration. Single external magnets can enhance magnetic nanoparticle delivery but are limited to superficial tumors due to the rapid decline in the magnetic field strength with distance. We previously showed that a 2-magnet device could extend targeting to greater tissue depths.

Robust, Scalable Microfluidic Manufacturing of RNA-Lipid Nanoparticles Using Immobilized Antifouling Lubricant Coating.

Despite the numerous advantages demonstrated by microfluidic mixing for RNA-loaded lipid nanoparticle (RNA-LNP) production over bulk methods, such as precise size control, homogeneous distributions, higher encapsulation efficiencies, and improved reproducibility, their translation from research to commercial manufacturing remains elusive. A persistent challenge hindering the adoption of microfluidics for LNP production is the fouling of device surfaces during prolonged operation, which significantly diminishes performance and reliability. The complexity of LNP constituents, including lipids, cholesterol, RNA, and solvent mixtures, makes it difficult to find a single coating that can prevent fouling.

High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level.

We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose ead-based igital Single Molecule-Single orting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches.

Mitigation of Device Heterogeneity in Graphene Hall Sensor Arrays Using Per-Element Backgate Tuning.

Graphene Hall-effect magnetic field sensors (GHSs) exhibit high performance comparable to state-of-the-art commercial Hall sensors made from III-V semiconductors. Graphene is also amenable to CMOS-compatible fabrication processes, making GHSs attractive candidates for implementing magnetic sensor arrays for imaging fields in biosensing and scanning probe applications. However, their practical appeal is limited by response heterogeneity and drift, arising from the high sensitivity of two-dimensional (2D) materials to local device imperfections.

Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells.

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells.

The next generation of hybrid microfluidic/integrated circuit chips: recent and upcoming advances in high-speed, high-throughput, and multifunctional lab-on-IC systems.

Since the field's inception, pioneers in microfluidics have made significant progress towards realizing complete lab-on-chip systems capable of sophisticated sample analysis and processing. One avenue towards this goal has been to join forces with the related field of microelectronics, using integrated circuits (ICs) to perform on-chip actuation and sensing. While early demonstrations focused on using microfluidic-IC hybrid chips to miniaturize benchtop instruments, steady advancements in the field have enabled a new generation of devices that expand past miniaturization into high-performance applications that would not be possible without IC hybrid integration.

Patterning Wettability on Solvent-Resistant Elastomers with High Spatial Resolution for Replica Mold Fabrication of Droplet Microfluidics.

Controlling the surface wetting properties of channels is crucial to the robust and reliable performance of microfluidic devices. Spatially patterned hydrophobic/hydrophilic microchannels have found utility across various applications, notably in the generation of higher-order emulsions. Unfortunately, the patterning of surface wettability currently requires multistep processes with limited spatial resolution, making it impractical for many applications.

Surface Topography-Adaptive Robotic Superstructures for Biofilm Removal and Pathogen Detection on Human Teeth.

The eradication of biofilms remains an unresolved challenge across disciplines. Furthermore, in biomedicine, the sampling of spatially heterogeneous biofilms is crucial for accurate pathogen detection and precise treatment of infection. However, current approaches are incapable of removing highly adhesive biostructures from topographically complex surfaces.

Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay.

Extracellular vesicles (EVs) have attracted enormous attention for their diagnostic and therapeutic potential. However, it has proven challenging to achieve the sensitivity to detect individual nanoscale EVs, the specificity to distinguish EV subpopulations, and a sufficient throughput to study EVs among an enormous background. To address this fundamental challenge, we developed a droplet-based optofluidic platform to quantify specific individual EV subpopulations at high throughput.

Plasma Neurofilament Light and Glial Fibrillary Acidic Protein Levels over Thirty Days in a Porcine Model of Traumatic Brain Injury.

To establish the clinical relevance of porcine model of traumatic brain injury (TBI) using the plasma biomarkers of injury with diffusion tensor imaging (DTI) over 30 days, we performed a randomized, blinded, pre-clinical trial using Yorkshire pigs weighing 7-10 kg. Twelve pigs were subjected to Sham injury ( = 5) by skin incision or TBI ( = 7) by controlled cortical impact. Blood samples were collected before the injury, then at approximately 5-day intervals until 30 days.

Scaling up the throughput of microfluidic droplet-based materials synthesis: A review of recent progress and outlook.

The last two decades have witnessed tremendous progress in the development of microfluidic chips that generate micrometer- and nanometer-scale materials. These chips allow precise control over composition, structure, and particle uniformity not achievable using conventional methods. These microfluidic-generated materials have demonstrated enormous potential for applications in medicine, agriculture, food processing, acoustic, and optical meta-materials, and more.