Nature Nanotechnology

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

Bottom-up manufacturing of structural DNA nanotechnology requires a long single-stranded DNA (ssDNA) scaffold and hundreds of short ([~]30 nt) ssDNA staples. However, scaling production remains bottlenecked by the high economic cost and environmental footprint of solid-phase chemical staple synthesis. To address these limitations, a phage-free, biological nanomanufacturing platform engineered in Escherichia coli is developed here. Two intracellular strategies for producing programmable ssDNA were systematically evaluated: retron-based multicopy ssDNA (msDNA) synthesis via the Ec67 system and plasmid-encoded rolling circle replication (RCR). While native structural topology constraints within the retron (msd) cassette limit its sequence-design flexibility, the alternative RCR-driven engine successfully decouples ssDNA replication from sequence secondary structures, enabling the synthesis of arbitrary staples. This RCR platform reliably generates long circular ssDNA (cssDNA) precursors of at least 1.8 kb with exceptional sequence fidelity (>99%). Integrating programmable BseGI cleavage sites allows targeted strand-selective enzymatic processing to cleanly release stoichiometric, origami-grade pools of 32-nt staple strands. Atomic force microscopy (AFM) confirms that these biologically produced staples successfully drive the high-fidelity self-assembly of complex DNA tiles and hollow tubules. Crucially, robust structural folding is demonstrated directly within crude, unpurified cellular lysates, establishing a green, cost-effective framework for the one-pot fabrication of advanced DNA-based nanomaterials.

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Atomically precise gold nanoclusters (AuNCs) are ultra-small particles composed of ten to hundreds gold atoms and exhibit unique photophysical properties. Significant progress has been made in tuning and extending their luminescence in the near-infrared window through the design of AuNC assemblies. Herein, we report a straightforward method for synthesizing highly pure, programmable DNA tetrahedra functionalized with a controlled number of AuNCs (from one up to four AuNCs). Using ligand exchange chemistry, AuNCs bearing a single grafted ssDNA onto them were produced. These constructs then served as building blocks for synthesizing tetrahedra through DNA hybridization. Products obtained at each stage of the synthesis were thoroughly characterized using a range of complementary technics. Notably, mass spectrometry in native mode provided novel insights into the accurate composition and stoichiometry of these architectures. This study paves the way for the synthesis and the characterization of a variety of new three-dimensional, DNA-guided AuNC assemblies that may serve as powerful theranostics and biophotonic tools.

Optical barcodes for pooled high-throughput screening must support large libraries while remaining decodable in a single imaging step. Existing approaches often trade design control for manufacturability: deterministic barcodes often require per-code redesign of particle fabrication, whereas stochastic combinatorial barcodes are difficult to generate as predefined batches. Here we introduce a chemically programmable barcoding architecture that decouples particle fabrication from barcode assignment. Using a contact-free multilaminar flow lithography platform with all-around three-dimensional sheathing, we continuously fabricate a universal hydrogel scaffold containing five spatially segregated DNA-addressable domains at rates >106 particles/h. Chosen barcode identities are subsequently written on demand onto the same template batch by domain-selective DNA hybridization. Single-domain measurements resolved 64 candidate optical states, indicating an experimentally informed theoretical upper bound of 645 {approx} 1.1 x 109 barcodes. We further implemented a predefined 59,049-code library by split-pool labeling, achieving an 88% recovery of decoded beads at a stringent posterior threshold (>0.95). After 11 days, >7,800 beads were correctly re-identified at >0.95 accuracy in matched fields of view. This strategy provides a highly scalable, chemically programmable route to build large, user-defined optical barcode libraries with single-image optical readout and longitudinal traceability.

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of [~]1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

Spatial and temporal organisation of membrane-associated components is fundamental to cellular signalling, yet remains difficult to engineer in minimal synthetic systems. In synthetic cells, DNA and RNA nanotechnology offer programmable molecular organisation at membranes, while in vitro transcription (IVT) enables gene expression-driven regulation. However, integrating these systems within cell-like compartments, such as giant unilamellar vesicles (GUVs), remains challenging due to undesirable interactions between transcription machinery and nucleic acid assemblies. Here, we present a modular strategy that couples in situ RNA production to dynamic DNA localisation at GUV synthetic cell membranes. RNA strands, transcribed within GUVs, function as linkers that recruit DNA-conjugated cargo to lipid membranes, enabling programmable spatial organisation. Using this framework, we achieved reversible membrane localisation through toehold-mediated strand displacement and RNase H-mediated degradation. This work establishes a gene expression-driven platform for programmable and dynamic control of membrane-associated components in synthetic cells, providing a foundation for constructing dynamic signalling assemblies and higher-order cellular behaviours. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/738173v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@9d8b5org.highwire.dtl.DTLVardef@726fd1org.highwire.dtl.DTLVardef@10b09bforg.highwire.dtl.DTLVardef@1ad19b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Encapsulins are prokaryotic self-assembling protein nanocages with promise as nanovaccine scaffolds. Their utility as modular platforms require tolerance to surface engineering, high-yield soluble production, formulation stability, and controlled antigen (co-)display. Herein, a previously uncharacterized encapsulin from Alkaliphilus metalliredigens is engineered into a SpyCatcher-decorated nanoscaffold (Am-S) that enables controlled surface display of SpyTagged antigens. Cryo-EM confirms that the native encapsulin forms a T = 1 icosahedral nanocage, and that C-terminal SpyCatcher fusion yields Am-S without compromising nanocage assembly, symmetry, or structural integrity. Notably, Am-S exhibits high-yield soluble production in Escherichia coli, remains monodisperse after freeze-thaw and extended storage, and supports efficient SpyTagged peptide conjugation for single- and multi-antigen display. As a proof-of-concept, Am-S is functionalized with Alzheimers disease-associated amyloid-{beta} and/or hyperphosphorylated tau epitopes to generate single-target nanocages displaying either antigen and dual-target mosaic nanocages co-displaying both. In mice, Am-S antigen display enhances antigen-specific IgG responses relative to free antigens and induces predominantly IgG1-biased humoral immunity. Mosaic nanocages elicit antibodies against both targets, with immune sera selectively recognizing amyloid-{beta}- and phosphorylated tau-associated pathology in ex vivo brain sections from Alzheimers disease mouse models. These findings position Am-S as a manufacturable scaffold for developing multi-targeting nanovaccines against complex diseases.

Human retina can achieve single-photon sensitivity through specialised photoreceptors that convert light into electrical signals via phototransduction. Among microbial light-sensitive proteins, proteorhodopsins stand out for their intrinsic light-driven ion transport and spectral tunability, making them promising candidates for bio-inspired photonic devices. A central challenge for acellular integration, however, is the fragility of most bacterial rhodopsins under extreme conditions. Here, we exploit the exceptional robustness of TARA76, a microbial rhodopsin that retains structural integrity even upon complete dehydration, to demonstrate its functional reconstitution in an artificial black lipid membrane within a biocompatible microfluidic platform. By recording light-induced ionic currents with picoampere sensitivity across a broad range of pH, illumination power, electrolyte composition, and applied voltages, we establish TARA76 as a high-performance photoelectric transducer in a fully acellular environment. Strikingly, we uncover a strong and previously unreported dependence of the photocurrent on Na+ ions, which appears to play a key structural and functional role in stabilising the proteins active conformation. Furthermore, we demonstrate that the orientation of TARA76 within the artificial membrane can be externally controlled by applying a defined electric field during bilayer formation, enabling deterministic tuning of photocurrent directionality. Together, these results establish a robust and miniaturisable bio-photonic platform with direct implications for quantum light sensing, neuromorphic bioelectronics, and next-generation artificial retinal interfaces.

In vivo genome editing with CRISPR-Cas9 ribonucleoproteins (RNPs) holds substantial therapeutic promise, yet rapid bloodstream clearance and the absence of delivery systems capable of systemic tumor targeting have hindered its clinical translation. Herein, a supramolecular ternary complex platform is reported in which Cas9/sgRNA RNPs are co-assembled with tannic acid (TA) and phenylboronic acid (PBA)-conjugated polymers through sequential self-assembly, producing [~]30 nm core-shell ternary complexes that protect RNPs from enzymatic degradation and dissociate selectively at endosomal pH. Upon intravenous administration in subcutaneous tumor-bearing mice, these ternary complexes exhibit prolonged blood circulation and preferential tumor accumulation, achieving 37.2% gene editing at tumor sites compared with only 1.5% for free RNPs. The platform successfully knocks out previously undruggable oncogenes including mutant KRAS and polo-like kinase 1 (PLK1), markedly suppressing tumor growth in vivo. By integrating sequential supramolecular self-assembly with stimuli-responsive cargo release, this strategy establishes a generalizable framework for systemically administered in vivo CRISPR therapeutics.

DNA nanotechnology is based on programmable base pairing, resulting in the precise construction of nanoscale structures. Sequence variability in DNA nanostructure self-assembly is achieved by the use of xeno nucleic acids, chemically modified bases and base analogs. The naturally occurring base inosine, while well studied in RNA editing, has not been used in the context of DNA nanotechnology. In this work, I demonstrate the use of inosine in DNA nanostructures, specifically by incorporating inosine within the duplex regions or junctions of a double crossover DNA motif. In strand displacement and competition assays, I show that canonical complements do not displace inosine containing strands post-assembly but dominate in product formation when competing with inosine containing strands during assembly. Finally, sticky ends with inosine base pairs enable the formation of rationally designed 3D DNA crystals based on the tensegrity triangle motif. Overall, this work shows that inosine is a useful addition to the library of sequence variations in DNA nanotechnology.

Controlling molecular transport across membranes is a defining feature of living cells, yet replicating this functionality in synthetic systems remains a major challenge. Self-inserting protein nanopores, such as -hemolysin (HL), offer a promising route towards programmable membrane permeability due to their robust assembly, compatibility with diverse membrane systems, and intrinsic permeability for diverse biomolecules. Here, we explored the use of chemically functionalized HL nanopores as tunable transport modules. To quantify translocation of peptide substrates across HL-containing membranes, we developed a high-throughput luminescence-based breakage-controlled assay using large unilamellar vesicles. With this assay we introduce a one-pot nanopore modification and strategy, compatible with scalable workflows. Electrophysiology and molecular simulations demonstrate that the introduction of cysteine residues at defined pore locations, combined with targeted chemical modification, enables controlled tuning of HL-nanopore selectivity based on peptide structure and charge. Together, these findings position engineered protein nanopores as versatile and responsive components for controlling membrane transport in synthetic biology.

Efficient and controllable delivery of genome-editing proteins remains a central challenge for therapeutic translation of gene-editing technologies. Extracellular vesicles (EVs) offer an attractive non-viral delivery modality due to their biocompatibility and large capacity for cytosolic cargo delivery. Yet, rational strategies to achieve controlled and programmable protein loading are still lacking. Here, we present NEO-TOP-EVs, an EV biogenesis-guided engineering platform that systematically integrates key features of three design principles inspired by vesicle formation: 1) PI(4,5)P2-mediated plasma membrane targeting, 2) ESCRT-dependent membrane scission, and 3) self-assembly-driven cargo clustering for enabling efficient encapsulation of genome-editing ribonucleoproteins. Together, the NEO design increased cargo incorporation and enhanced functional delivery of gene editing modalities under particle-normalized conditions. Using NEO-TOP-EVs, we achieve efficient delivery of Cas9 and adenine base editor ribonucleoproteins without nucleic acid templates. In an in vitro proof-of-concept, delivery of an adenine base editor targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) induces efficient splice-site disruption, resulting in reduced PCSK9 expression and enhanced LDL receptor activity. Proof-of-concept in vivo experiments provide preliminary evidence of functional Cre protein delivery to the liver. Together, these findings establish NEO-TOP-EVs as a modular platform for protein-based genome editing, demonstrating how biogenesis-informed EV engineering yields functional protein delivery at levels relevant to therapeutic development.

Negatively charged DNA nanostructures, such as tetrahedral nanocages, are internalized by cells despite the electrostatic repulsion from the anionic cell membrane, and, paradoxically, cancer cells, which carry intrinsically higher negative charge due to overexpression of sialic acids on their cell surface, show markedly higher uptake than normal cells. This contradiction exposes a fundamental gap in our understanding of how these anionic nanostructures overcome this repulsion. Using chemical modulation of cell-surface sialylation in RPE1 cells to create three groups with altered sialylation levels, together with inhibitor-based dissection of endocytic pathways, we demonstrate that an increase in cell surface sialylation governs the uptake of DNA tetrahedra not through electrostatics but by structurally remodeling the cell membrane via rearrangement of the GM1 lipid raft microdomain, recruiting caveolae-mediated endocytosis as an additional pathway alongside clathrin-mediated endocytosis, thereby increasing the intake of the nanostructure. These findings reframe tumor hyper-sialylation as a determinant of the uptake of anionic nanostructures, such as DNA tetrahedra, and as a targetable parameter for rational optimization of DNA-based nanotherapeutics against cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/722926v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10eede7org.highwire.dtl.DTLVardef@124dd56org.highwire.dtl.DTLVardef@13f5355org.highwire.dtl.DTLVardef@780ecf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Systemic delivery of nucleic acids to the central nervous system (CNS) remains fundamentally limited by the blood-brain barrier (BBB) and the lack of cell-type specificity. Inspired by endogenous high-density lipoproteins (HDLs) that naturally traverse the BBB, we engineered a library of 112 HDL-inspired lipid nanoparticles (HLNPs) to deliberately program protein corona composition and direct nanoparticle trafficking across the BBB. High-throughput in vivo screening identified HLNPs with robust brain accumulation and preferential enrichment within distinct neural cell populations. Quantitative proteomic analysis revealed that elevated apolipoprotein A-I/apolipoprotein E and vitronectin/apolipoprotein E ratios correlate with enhanced BBB transport. We further demonstrated that neuron-targeted HLNPs delivering PTEN siRNA produced functional recovery and lesion reduction in a murine traumatic brain injury model, while microglia-targeted HLNPs carrying interleukin-10 mRNA potently suppressed neuroinflammation. Together, these findings establish HLNPs as a versatile RNA delivery platform that leverages protein corona programming for enhanced brain delivery and cell-type-selective targeting.

Oligonucleotide therapeutics hold transformative potential, yet their clinical translation is hindered by delivery barriers, including rapid renal/hepatic clearance and poor organ specificity. Bottlebrush polymers conjugates have emerged as a promising vector to address these limitations, but conventional architectures with uniform backbones can only achieve an unmodifiable, rigid biodistribution profile. Here, we report a library of sequence-defined "digital" bottlebrush polymers, precisely engineered with controlled placements of chemical motifs that modify physiochemical properties - including lipids, cholesterol, and cationic groups - along a polyphosphodiester backbone. Systematic evaluation of the digital bottlebrush polymer library reveals distinct structure-property relationships and enables organ-biased systemic delivery to several traditionally difficult-to-reach tissues, including muscle and skin. In a mouse model of rheumatoid arthritis, a single dose of a spleen-homing polymer-conjugated antisense oligonucleotide targeting TNF- achieves potent knockdown and drives full functional recovery. These findings establish a versatile design framework for tailoring bottlebrush polymers to specific therapeutic applications.

DNA-barcoded antibodies are central to a broad range of spatial and dissociative assays and applications, including multiplexed imaging, single-cell profiling, and proximity detection. Direct modification of primary antibodies with defined DNA barcodes enables flexible panel design for multiplexed labeling of proteins. However, conventional antibody-oligonucleotide conjugation methods are inefficient, low-throughput, and prone to batch variability, limiting the reliable generation of orthogonally barcoded antibody panels. These challenges are particularly acute during initial panel development, where lengthy protocols, conjugation failures, large antibody input requirements, and the need for custom antibody formulations increase experimental cost and effort. Site-specific conjugation strategies based on antibody Fc-domain binders offer a promising alternative. We streamlined this foundational approach to establish its compatibility with multiplexed imaging in cells and tissues; however, generating a full barcoded library is still resource-intensive. This is because every unique DNA barcode must first be chemically linked to a separate binder before it can be attached to an antibody. To overcome the prominent bottleneck of rapidly and reliably generating DNA-barcoded antibody panels, we introduce the universal oligo adapter (UnO) strategy. UnO fundamentally changes the workflow from barcode-specific conjugation to universal barcode conversion. We build on the established photoreactive protein G binder and combine it with a universal oligonucleotide carrying a second ultrafast photocrosslinking group, 3-cyanovinylcarbazole (cnvK). This creates a dual-functional adapter: one photoreactive group enables covalent attachment to the antibody, while the cnvK-containing universal oligo simultaneously captures a user-defined DNA barcode through hybridization and UV crosslinking in a single step. Rather than preparing separate conjugation reactions for dozens of barcodes, UnO acts as a single reagent that covalently couples any desired barcode onto small quantities of off-the-shelf primary antibodies in minutes. We validate the generalizability and modularity of this approach across subcellular Immuno-SABER and tissue-based CODEX workflows for multiplex immunostaining. By converting antibody barcoding into a modular, one-step nucleic-acid adapter workflow, UnO reduces the cost, time, and complexity of generating DNA-barcoded antibody panels and provides an efficient, accessible solution to a central bottleneck in DNA-enabled multiplexed protein detection.

DNA-programmed assembly of plasmonic nanostructures provides a powerful route to couple molecular recognition with optical signal generation. Here, we report the sequence-specific assembly of DNA-functionalized gold nanorods using a sesame allergen-derived DNA biomarker as a molecular bridge. Target-induced assembly produces concentration-dependent assembly growth, plasmon coupling, and distinct assembly kinetics that are readily monitored by absorption spectroscopy, enabling label-free detection of the target DNA in the nanomolar concentration range. The assembled nanorods further produce strong surface-enhanced Raman scattering (SERS) signals arising from plasmonic coupling within the assemblies, extending detection to the picomolar regime without the use of Raman reporters. Quantitative analysis reveals that both the extent and rate of assembly formation are governed by target DNA concentration. These results establish a direct relationship between molecular recognition, assembly growth, plasmonic coupling, and spectroscopic response, highlighting DNA-programmed gold nanorod assembly as a versatile platform for investigating hybridization-driven plasmonic self-assembly and nucleic acid detection. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/732610v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@6a8f42org.highwire.dtl.DTLVardef@1e36b9corg.highwire.dtl.DTLVardef@1ade546org.highwire.dtl.DTLVardef@1a787bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.

The accurate quantification of biological nanoparticles, such as small extracellular vesicles (sEVs), is fundamentally hindered by a resolution-coincidence trade-off in digital assays. While physical confinement can isolate single particles, conventional optical readouts remain diffraction-limited, causing multi-particle occupancy to be miscounted as single events and thereby restricting the analytical dynamic range. Here, we report a nanoplasmonic platform that overcomes this limit by introducing a geometry-defined interface that uniquely unifies nanoscale compartmentalisation and near-field-assisted super-resolution imaging. Utilising a gold plasmonic nanohole array, the strict geometric periodicity of the lattice simultaneously serves as a template for single-vesicle confinement and a deterministic grid that generates an array of localised surface plasmon resonance near-field hotspots. This position-deterministic illumination pattern imposes known geometric priors on the excitation field, shifting high-spatial-frequency information into the detectable bandwidth to achieve sub-100 nm lateral resolution. This dual-purpose geometric determinism enables high-fidelity digital readout of individual vesicles with significantly fewer sub-images than stochastic, speckle-based metasurface structured illumination microscopy approaches. The assay achieves an analytical limit of detection of 143 sEVs/{micro}L, matching the performance of state-of-the-art single-EV counting technologies. It successfully differentiates distinct sEV subpopulations based on surface biomarker expression, establishing a clear pathway for future clinical liquid biopsy applications. By replacing stochastic loading and illumination with geometric design, this work establishes a robust framework for precise vesicle interrogation with broad implications for emerging translational applications and fundamental biology.