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Extracellular vesicles (EVs) – nanoscale membranous particles that carry multiple proteins and nucleic acid cargoes from their mother cells of origin into circulation – have enormous potential as biomarkers. However, devices appropriately scaled to the nanoscale to match the size of EVs (30–200 nm) have orders of magnitude too low throughput to process clinical samples (10¹²EVs mL⁻¹in serum). To address this challenge, we develop a novel approach that incorporates billions of nanomagnetic sorters that act in parallel to precisely isolate sparse EVs based on immunomagnetic labeling directly from clinical samples at flow rates billions of times greater than that of a single nanofluidic device. To fabricate these chips, the ferromagnetic metals are electro‐deposited into a self‐assembled microlattice, achieving >10⁹nanoscale magnetophoretic sorting devices in a 3D postage stamp‐sized lattice with >70x magnetic traps and >20x enrichment of magnetic nanoparticles versus our previous work. The immunomagnetically labeled EVs are isolated and achieve a ≈100% increase in yield as well as increased purity compared to conventional methods. Building on the proof‐of‐concept demonstrations in this manuscript, this new approach has the potential to enhance the future clinical translation of EV biomarkers by enabling rapid, sensitive, and specific isolation of EV subpopulations from clinical samples.

 

Abstract Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys302 and Glu267 resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD+ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD+ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe169 with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae.