exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer.

OBJECTIVE: Surveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions. DESIGN: EVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study. RESULTS: ExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (p<0.01, n=157). An independent validation in a phase 2 biomarker case-control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93. CONCLUSION: These findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

Broken flow symmetry explains the dynamics of small particles in deterministic lateral displacement arrays.

Deterministic lateral displacement (DLD) is a technique for size fractionation of particles in continuous flow that has shown great potential for biological applications. Several theoretical models have been proposed, but experimental evidence has demonstrated that a rich class of intermediate migration behavior exists, which is not predicted. We present a unified theoretical framework to infer the path of particles in the whole array on the basis of trajectories in a unit cell. This framework explains many of the unexpected particle trajectories reported and can be used to design arrays for even nanoscale particle fractionation. We performed experiments that verify these predictions and used our model to develop a condenser array that achieves full particle separation with a single fluidic input.

Deterministic Lateral Displacement: Challenges and Perspectives.

The advent of microfluidics in the 1990s promised a revolution in multiple industries from healthcare to chemical processing. Deterministic lateral displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. Deterministic lateral displacement is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations. Despite wide use and in-depth study, DLD has not yet been fully elucidated or optimized, with different approaches to the same problem yielding varying results. We endeavor here to provide up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges with DLD as well as describe experimental and modeling opportunities. Because these challenges and opportunities arise from constraints on hydrodynamics, fabrication, and operation at the micro- and nanoscale, we expect this Perspective to serve as a guide for the broader micro- and nanofluidic community to identify and to address open questions in the field.

Gel-on-a-chip: continuous, velocity-dependent DNA separation using nanoscale lateral displacement.

We studied the trajectories of polymers being advected while diffusing in a pressure driven flow along a periodic pillar nanostructure known as nanoscale deterministic lateral displacement (nanoDLD) array. We found that polymers follow different trajectories depending on their length, flow velocity and pillar array geometry, demonstrating that nanoDLD devices can be used as a continuous polymer fractionation tool. As a model system, we used double-stranded DNA (dsDNA) with various contour lengths and demonstrated that dsDNA in the range of 100-10 000 base pairs (bp) can be separated with a size-selective resolution of 200 bp. In contrast to spherical colloids, a polymer elongates by shear flow and the angle of polymer trajectories with respect to the mean flow direction decreases as the mean flow velocity increases. We developed a phenomenological model that explains the qualitative dependence of the polymer trajectories on the gap size and on the flow velocity. Using this model, we found the optimal separation conditions for dsDNA of different sizes and demonstrated the separation and extraction of dsDNA fragments with over 75% recovery and 3-fold concentration. Importantly, this velocity dependence provides a means of fine-tuning the separation efficiency and resolution, independent of the nanoDLD pillar geometry.

Integrated nanoscale deterministic lateral displacement arrays for separation of extracellular vesicles from clinically-relevant volumes of biological samples.

Extracellular vesicles (EVs) offer many opportunities in early-stage disease diagnosis, treatment monitoring, and precision therapy owing to their high abundance in bodily fluids, accessibility from liquid biopsy, and presence of nucleic acid and protein cargo from their cell of origin. Despite their growing promise, isolation of EVs for analysis remains a labor-intensive and time-consuming challenge given their nanoscale dimensions (30-200 nm) and low buoyant density. Here, we report a simple, size-based EV separation technology that integrates 1024 nanoscale deterministic lateral displacement (nanoDLD) arrays on a single chip capable of parallel processing sample fluids at rates of up to 900 µL h-1. Benchmarking the nanoDLD chip against commonly used EV isolation technologies, including ultracentrifugation (UC), UC plus density gradient, qEV size-exclusion chromatography (Izon Science), and the exoEasy Maxi Kit (QIAGEN), we demonstrate a superior yield of ∼50% for both serum and urine samples, representing the ability to use smaller input volumes to achieve the same number of isolated EVs, and a concentration factor enhancement of up to ∼3× for both sample types, adjustable to ∼60× for urine through judicious design. Further, RNA sequencing was carried out on nanoDLD- and UC-isolated EVs from prostate cancer (PCa) patient serum samples, resulting in a higher gene expression correlation between replicates for nanoDLD-isolated EVs with enriched miRNA, decreased rRNA, and the ability to detect previously reported RNA indicators of aggressive PCa. Taken together, these results suggest nanoDLD as a promising alternative technology for fast, reproducible, and automatable EV-isolation.

Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20†nm.

Deterministic lateral displacement (DLD) pillar arrays are an efficient technology to sort, separate and enrich micrometre-scale particles, which include parasites, bacteria, blood cells and circulating tumour cells in blood. However, this technology has not been translated to the true nanoscale, where it could function on biocolloids, such as exosomes. Exosomes, a key target of 'liquid biopsies', are secreted by cells and contain nucleic acid and protein information about their originating tissue. One challenge in the study of exosome biology is to sort exosomes by size and surface markers. We use manufacturable silicon processes to produce nanoscale DLD (nano-DLD) arrays of uniform gap sizes ranging from 25 to 235 nm. We show that at low Péclet (Pe) numbers, at which diffusion and deterministic displacement compete, nano-DLD arrays separate particles between 20 to 110 nm based on size with sharp resolution. Further, we demonstrate the size-based displacement of exosomes, and so open up the potential for on-chip sorting and quantification of these important biocolloids.

Hydrodynamics of diamond-shaped gradient nanopillar arrays for effective DNA translocation into nanochannels.

Effective DNA translocation into nanochannels is critical for advancing genome mapping and future single-molecule DNA sequencing technologies. We present the design and hydrodynamic study of a diamond-shaped gradient pillar array connected to nanochannels for enhancing the success of DNA translocation events. Single-molecule fluorescence imaging is utilized to interrogate the hydrodynamic interactions of the DNA with this unique structure, evaluate key DNA translocation parameters, including speed, extension, and translocation time, and provide a detailed mapping of the translocation events in nanopillar arrays coupled with 10 and 50 µm long channels. Our analysis reveals the important roles of diamond-shaped nanopillars in guiding DNA into as small as 30 nm channels with minimized clogging, stretching DNA to nearly 100% of their dyed contour length, inducing location-specific straddling of DNA at nanopillar interfaces, and modulating DNA speeds by pillar geometries. Importantly, all critical features down to 30 nm wide nanochannels are defined using standard photolithography and fabrication processes, a feat aligned with the requirement of high-volume, low-cost production.

Triplet exciton dissociation in singlet exciton fission photovoltaics.

Triplet exciton dissociation in singlet exciton fission devices with three classes of acceptors are characterized: fullerenes, perylene diimides, and PbS and PbSe colloidal nanocrystals. Using photocurrent spectroscopy and a magnetic field probe it is found that colloidal PbSe nanocrystals are the most promising acceptors, capable of efficient triplet exciton dissociation and long wavelength absorption.

Divalent metal nanoparticles.

Nanoparticles can be used as the building blocks for materials such as supracrystals or ionic liquids. However, they lack the ability to bond along specific directions as atoms and molecules do. We report a simple method to place target molecules specifically at two diametrically opposed positions in the molecular coating of metal nanoparticles. The approach is based on the functionalization of the polar singularities that must form when a curved surface is coated with ordered monolayers, such as a phase-separated mixture of ligands. The molecules placed at these polar defects have been used as chemical handles to form nanoparticle chains that in turn can generate self-standing films.