ACS Nano

Single-walled carbon nanotubes (SWCNTs) show promise for optical biosensing, imaging, and drug delivery, but turning them into safe, precision nanomedicine tools requires understanding how nanotube surface chemistry dictates recognition and processing by cells. Like other nanomaterials, carbon nanotubes acquire a biomolecular corona on contact with biological fluids, and corona identity is increasingly recognized as central to sensor performance and drug delivery efficacy. However, whether corona identity also governs the intracellular fate of carbon nanotubes remains largely unknown. Here, we show that the single-stranded DNA wrapping of (6,5)-enriched single-walled carbon nanotubes reprograms their protein corona, intracellular trafficking, and macrophage response. By profiling (AT)15, (GT)15, and (CT)15 wrapped SWCNTs, we show that the wrapping sequence programs both the protein corona and the resulting proteostatic stress on macrophages. Photoluminescence imaging and confocal Raman measurements reported that (AT)15 is internalized the most yet leaves the proteome and nanotube structure largely undisturbed, whereas (CT)15, taken up the least, undergoes the most aggressive intracellular degradation and drives the highest oxidative and proteostatic stress. Corona proteomics indicated that all three tested nanotubes form coronas with distinct functional identities that are responsible for divergent intracellular routes. Time-resolved intracellular proteomics combined with functional assays resolved how the host cell reorganizes its biomolecular complexity over time, including oxidative outputs, aside from a sequence-independent core response involving particle engagement, phagosomal sorting, and lysosomal processing. These findings provide mechanistic insight into nanomaterial-cell interactions and the wrapping sequence as a tunable, nucleotide-level design handle for controlling the intracellular fate of carbon nanomaterials, with potential implications for safe and effective nanomedicine platforms.

Cell membrane-coated nanoparticles (CNPs) are a potentially transformative biomimetic targeted-delivery platform, which can engage therapeutic targets through source-cell membrane protein functions or homotypic interactions. However, their therapeutic development is limited by the current inability to accurately resolve membrane coating completeness, and CNP particle-to-particle variation. Here, we introduce a STochastic Optical Reconstruction Microscopy (STORM)-based approach that resolves population composition and quantifies membrane coating coverage at the single-particle level. Applying it to dendritic cell membrane- and HeLa cell membrane-coated nanoparticles (DCmPs and HeLamPs), we revealed substantial heterogeneity within both particle types and a cell-type dependence of coating outcomes, corroborated by confocal imaging and flow cytometry. We also quantified membrane protein composition, including peptide major histocompatibility complex class I (pMHC-I) among DCmPs, and confirmed that DCmPs preferentially engaged antigen-specific T cells in coculture. This single-particle framework establishes a quantitative standard for CNP characterization, providing a foundation for quality control and rational design.

Existing biological nanopores are often too narrow to accommodate large proteins and biomolecular complexes in their native folded states, precluding the analysis of megadalton assemblies. Here, we report the self-assembly of the cholesterol-dependent cytolysin Perfringolysin O (PFO) into stable transmembrane nanopores composed of 46 {+/-} 9 monomers, with an inner pore diameter of 28.5 {+/-} 5.6 nm and a length of 9.8 nm. We demonstrate that despite the large size of the pore assembly, PFO pores exhibit a stable open-pore current with a high signal-to-noise ratio, making them suitable for resistive-pulse recordings. Moreover, PFO nanopores enable accurate, calibration-free, and label-free sizing of individual proteins, multi-protein complexes, and viral particles across a large molecular-weight range spanning 50 kDa to 3.4 MDa. The exceptionally large diameter of these pores enables, for the first time, resistive-pulse-based characterization of intact virus particles and ribosomes with a biological nanopore. Specifically, we determined the volume, shape, and diameter of complete capsids of recombinant adeno-associated virus serotype 2 (rAAV2) as well as capsid fragments. Finally, simultaneous analysis of molecular volume and shape resolved intact 70S ribosomes from their dissociated 30S and 50S subunits in a mixture. By extending biological nanopore sensing to single-particle characterization of large protein complexes well beyond the reach of existing pores, this approach introduces PFO nanopores as a versatile platform for label-free, single-particle analysis in solution.

Viral diagnostics during early phases of infection are often limited by target scarcity and the deployment tempo. We significantly advance both quantitative accuracy and diagnostic throughput of viral agglutination assays with Immuno-Janus Particle (IJP) aggregation behavior that "flicker" stochastically with size-dependent statistics. By scrutinizing microscale blinking patterns of time series fluorescent videos, we decipher Brownian dynamics of individual IJP-Virus conjugates and IJP aggregates via windowed Ito stochastic analysis (termed the Culsans method). High-frequency rotational fluctuation is deconvolved from corrupting drifts caused by gravitational sedimentation and Brownian translational motion. This methodology enables a non-linear mapping of angular positions of detected IJPs and IJP aggregates to extract rotational diffusivity (Dr) (and subsequently overall construct size) with superior linearity (R2[≥]0.85). The aggregation behavior exhibits a maximum when the IJP and viral particle concentrations are equal. The virion-bridged IJP-IJP conjugates significantly shift the detectable hydrodynamic diameter in the Poisson limit of reduced virus concentration with respect to IJPs, pushing the limit of detection (LOD) to 103 - 104 virions per mL in untreated human plasma. This tunable platform offers a rapid, low-volume, and scalable alternative to lab-based RT-PCR, bridging the gap between virion sensitivity and field-readiness.

Rapid DNA transport limits the temporal resolution of solid-state nanopore sensing. Here we show that breaking pore symmetry through geometric inclination produces exceptionally prolonged DNA residence without surface functionalization or external trapping. For 500-base-pair double-stranded (ds) DNA ,13 nm diameter SiNx pores in tilted membranes exhibit a distinct long-lived population with a mean dwell time of 11.1 seconds at 600 mV bias, exceeding previously reported dsDNA dwell times by at-least 4 orders of magnitude. Controlled pore tilting reproduces the population of prolonged events, whose prevalence and duration depend on ionic strength. TEM-EELS based thickness measurements exclude substantial membrane-thickness differences, while continuum and atomistic simulations support a mechanism coupling electric-field redistribution to off-axis DNA motion, increased pore-wall contact and suppressed axial mobility. These findings establish geometric inclination as a passive design variable for engineering molecular residence in solid-state nanopores and provide a route towards longer observation windows for single-molecule sensing.

Nanomedicines that perform well in conventional cell cultures often fail to translate into patients, in part due to the inability to reproduce the protein corona, and hence the biological identity, that nanoparticles acquire within the tumor microenvironment (TME). Here, we establish patient-derived colonic organoids (PDCOs) as a platform for profiling nano-bio interactions under physiologically tumor-relevant conditions using graphene quantum dots (GQD) and Silicon Quantum Dots (SiQD) as material-distinct probes. By combining spectral flow cytometry, confocal imaging, dynamic light scattering, and multi-spectral dimension-reduction analysis of single-cell uptake, we resolve how nanoparticle identity, uptake, and intracellular fate diverge between conventional culture and patient tissue. Conventional two-dimensional culture overestimated internalization by two-fold relative to ex vivo PDCOs, while TME-specific proteins directed nanoparticles to distinct organoid subpopulations. Exploiting this, we engineer the protein corona with tumor-specific proteins to redirect SiQDs to chemoresistant cells, from 3.8% to 70%, an 18-fold increase in targeted delivery to the cells that drive therapy resistance. Internalization was also model-dependent. PDCOs and fibroblasts favored clathrin/dynamin (DNM1)-mediated endocytosis into lysosomes, whereas cancer cells favored caveolae (CAV1)-mediated entry that bypasses lysosomal degradation. This distinction determines whether a nanocarrier is degraded or delivered intact. Collectively, these findings turn the protein corona into a programmable design tool and establish PDCOs as a next-generation platform for precision nano-oncology.

Solid-state nanopores offer a versatile platform for single-molecule sensing owing to their mechanical robustness, tuneable geometry, and compatibility with scalable fabrication. Here, we present a pyramidal silicon nanopore with a 40 nm sensing aperture for label-free characterisation of protein molecules by resistive pulse sensing. The nanopore operates stably over transmembrane voltages ranging from -2 to +2 V and across a broad range of electrolyte concentrations, enabling analysis under diverse experimental conditions. As molecules traverse the confined sensing region, transient ionic current modulations are generated that reflect their excluded volume and molecular geometry. Using this approach, we characterise unlabelled Tau species spanning monomeric proteins, intermediate aggregates, and mature fibrillar assemblies. Analysis of the resulting current signatures, together with simplified geometrical models, enables reconstruction of molecular dimensions and discrimination of distinct Tau populations based on their electrical fingerprints. These results demonstrate that pyramidal silicon nanopores provide a sensitive and scalable platform for label-free monitoring of structurally heterogeneous protein aggregation and establish a framework for investigating protein aggregation using solid-state nanopore sensing.

Virus-like particles (VLPs) are non-infectious, multiprotein nanostructures that mimic the architecture of viruses while lacking genetic material. Their structural versatility and safety profile make them promising platforms for biomedical applications, including vaccine development, targeted drug delivery, and diagnostics. Among various functionalization strategies, click-chemistry has emerged as a precise and efficient method for attaching bioactive molecules to VLP surfaces. In this study, we report the production and functionalization of VLPs derived from the feline panleukopenia virus (FPV), a parvovirus closely related to the well-characterized canine parvovirus (CPV). We demonstrate successful expression, assembly, and purification of FPV VLPs, followed by their modification with fluorophores, peptides, oligonucleotides, and proteins using copper-free click-chemistry. Furthermore, using cryo-EM, we structurally characterize FPV VLP functionalized with antigenic peptides, providing detailed insights for further development of such vaccine prototype. Finally, we demonstrate that our FPV VLP specifically transduce feline and human cells expressing transferrin receptor type 1 (TfR), thus validating their potential as a unique platform for future nanotechnological and therapeutic applications.

Selectively perturbing pathogenic membranes while preserving host-cell integrity is a fundamental challenge in the design of membrane-active biomaterials. This challenge is particularly acute for enveloped viruses because their lipid membranes are derived from host cells and therefore share many of the same molecular constituents. Here, we demonstrate that engineered nanochirality provides a structural parameter for distinguishing virus-organized membrane nanodomains from host-cell membranes. Histidine-functionalized graphene quantum dots (His-GQDs) form enantiomeric nanoscale structures with distinct interfacial topologies despite closely matched size, composition, and surface charge. D-His-GQDs preferentially interact with cholesterol-rich ordered membrane nanodomains associated with S-acylated coronavirus Spike, penetrate more deeply into raft-like lipid bilayers, and induce lipid disordering, membrane leakage, and viral-envelope disruption. Reducing Spike S-acylation or depleting membrane cholesterol attenuated this stereoselective interaction, while molecular simulations revealed asymmetric lipid organization surrounding S-acylated Spike and preferential insertion of D-His-GQDs into the ordered membrane environment. Functionally, D-His-GQDs directly inactivated human coronavirus OC43 (HCoV-OC43), inhibited infection with an EC50 of 0.92 g/mL and exhibited a selectivity index of 361. Stereoselective antiviral activity extended to human cytomegalovirus, demonstrating activity across distinct enveloped-virus families, and intranasal D-His-GQDs protected HCoV-OC43-challenged mice under both prophylactic and early post-exposure regimens. Overall, these findings establish structural nanochirality as a biomaterial design parameter for recognizing higher-order membrane organization and converting virus-associated membrane states into selectively addressable therapeutic vulnerabilities.

The nuclear pore complex (NPC) is a massive protein system that controls all nucleocytoplasmic transport via a dynamic network of intrinsically disordered proteins called FG-Nups. We developed an NPC-mimicking DNA origami nanostructure that self-assembles into a tetrameric, octagonal nanopore with an inner diameter that, through addition of DNA oligonucleotides, can be tuned in a user-defined fashion from 57 to 66 nm - mimicking the contracted and dilated states of NPCs. We demonstrated that these origami nanopores can dilate within minutes and recontract at slower timescales. Both contracted and dilated pores can be inserted into lipid bilayers, where they maintain their conformation and allow transmembrane transport of fluorescent proteins. With outer diameters up to 87 nm, these are, to our knowledge, the largest DNA origami nanopores inserted into lipid membranes. Functionalization of the pores with the FG-Nup Nsp1 reduces non-specific cargo diffusion across the lipid membrane while facilitating translocation of the transport receptor Kap95, demonstrating transport selectivity much like biological NPCs. The work establishes a size-tunable platform to dissect nuclear transport mechanisms, with potential applications for engineering programmable artificial channels.

Circulating microRNAs (miRNAs) have emerged as promising liquid biopsy markers; however, their clinical translation is restricted by low abundance, biological heterogeneity, and the complexity of multiplex profiling. Herein, we develop a targeted cycling-mediated duplex-specific nuclease (DSN)-nanozyme signal amplification platform (TC-DSN-Nano) that establishes a compartmentalized catalytic transduction strategy for multiplexed miRNA analysis. By encapsulating gold nanozymes within liposomal nanoreactors, this platform spatially separates molecular recognition from catalytic signal generation, preserving nanozyme activity during sensor construction. Upon target miRNA recognition, DSN-mediated recycling induces regenerative release of nanozyme-loaded liposomes from magnetic substrates, converting individual miRNA molecules into amplified catalytic outputs without conventional nucleic acid amplification. The platform enables programmable profiling of six prostate cancer (PCa) associated miRNAs in serum samples. Furthermore, machine learning-assisted integration of multiplexed miRNA signatures enables accurate discrimination among healthy donors, nonmetastatic PCa patients, and metastatic PCa patients, achieving an overall classification accuracy of 90.5%. This work establishes a general compartmentalized nanozyme transduction framework that integrates molecular recycling, catalytic amplification, and computational analysis for liquid biopsy-based cancer stratification.

Biomarker analysis requires detecting analyte classes that span nucleic acids, proteins, small molecules, and metabolites, yet testing remains fragmented across target-specific assays and instruments. Here we report a molecular information-transduction strategy that converts target recognition across molecular classes into a common, error-tolerant DNA code readable by nanopore sequencing. Target recognition triggers a hybridization chain reaction that generates concatemers containing periodically repeated 10-nucleotide target-specific barcodes. A matched-filter decoder exploits this periodicity and the linear scaling of read length with match count to reject spurious matches by two to three orders of magnitude. Multi-class detection is demonstrated for a small molecule (ATP), two cardiovascular-associated microRNAs and thrombin in singleplex and multiplexed assays. By separating molecular recognition from sequence readout, this architecture provides a modular framework for converting heterogeneous analytes into a shared, redundancy-encoded signal for high-fidelity molecular sensing.

Our environment has become progressively contaminated with non-biological materials, especially synthetic carbon polymers, plastics. Unsurprisingly some of these make it into the human body as detected by chemical analyses, yet it is unknown whether they cause harm or are innocent bystanders. Building on our observations of glossy deposits and unusual non-biological fluorescent particles in blood vessels, we aimed to determine whether these objects represented plastics. To do this we prepared plastics-enriched pellets from brain and subjected them to pyrolysis gas-chromatography/mass spectroscopy (py-GC/MS), electron microscopy and confocal laser scanning microscopy. Py-GC/MS confirmed the presence of 10 different plastics. Contents of pellets were examined by thin-section EM. We then used laser scanning confocal microscopy to acquire hyperspectral profiles of each type of plastic in suspensions. We found fluorescent particles in all pellets from brains. All 12 plastics in the calibration standard fluoresced. We obtained hyperspectral profiles of single species plastics from industry: Polyethylene, polypropylene and polystyrene. Controls included chemical analysis of brain storage buffer, which had no detectable plastics; and imaging water only and areas on slides lacking tissue. Abundant particles with emission profiles similar to polyethylene and polypropylene decorated the walls of both arterioles and venules. These particles were coincident with glossy deposits we first observed in white matter, suggesting that both features represent plastics. In summary our results demonstrate that synthetic polymers exhibit detectable fluorescence, and particles with similar spectral properties are visible in histologic sections. This opens the door to investigating correlations between plastics and pathology.

Expansion microscopy (ExM) physically magnifies preserved biological specimens by using a specimen-permeating hydrogel to pull biomolecules, such as proteins, apart from each other. ExM protocols exhibit a tension between the need to initially fix proteins to each other during sample preservation, to retain proteins and their nanoscale organization, and the need to separate proteins from each other isotropically, to achieve physical magnification. We here propose to address this tension by fixing proteins directly to the hydrogel network itself, which then serves as a common substrate for both initial fixation and final separation. We equip proteins with chemical handles, then form a specimen-permeating hydrogel that binds the handles, in a ~15-minute chemical process conducted entirely at -20oC. This network fixation ExM (nfExM) procedure enables excellent epitope retention and nanoscale organization preservation, in cultured cells. nfExM may help support ultrastructural investigations of protein complexes and interactions.

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

A broad-spectrum antiviral is required to enable rapid intervention against current, newly emerging and mutating viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), Sindbis virus (SINV) and chikungunya virus (CHIKV) all engage negatively charged moieties on host cells, such as heparan sulfate (HS) and sialic acid, to facilitate attachment for entry and infection. We designed HS-mimetic nanoparticles in the form of polymer vesicles (polymersomes) by blending membrane-forming and sulfonated copolymers. These biocompatible polymersomes successfully inhibited host cell infection of HS-dependent SARS-CoV-2 and HS-independent IAV in vitro with sub-nanomolar potency. Multivalent interaction of several virus ligands with a single polymersome as quantified by fluorescence correlation spectroscopy likely contributes to their potency. The sulfonated polymersomes also functioned in vivo as demonstrated by a reduction in the spread of SINV, an alphavirus model for CHIKV, in zebrafish embryos, which led to a significantly increased survival. Hence, our polymersomes could form the basis for an urgently required multifunctional broad-spectrum antiviral nanomedicine.

Biological processes are often regulated through reactions involving supramolecular protein complexes organized with nanoscale precision, such as the apoptosome that activates procaspase-9 through defined stoichiometry to induce apoptosis. Here we report designer CASCAD (CARD Assembly on SCAffold DNA), a self-assembled DNA nanoplatform that spatially organizes caspase recruitment domains (CARDs), exhibiting catalytic intracellular caspase-9 activation. By functioning like a natural apoptosome, CASCAD enhances caspase-9 oligomerization and promotes valency-dependent enzymatic activation. Further, after incorporating targeting aptamers and membrane-interacting peptides for cellular uptake and cytosolic delivery, CASCAD has successfully reconstituted apoptosome function and downstream apoptotic signaling in in-vitro 2D and 3D cultures. Our molecular design paradigm establishes CASCAD as versatile and programmable DNA nanomaterial for reconstructing signaling pathways driven by diverse supramolecular complexes, including inflammasomes and myddosomes, providing a general chemical framework for engineering cellular signaling and directing cellular function and fate.

Subcellular thermal engineering provides a powerful approach for investigating and manipulating biological processes. However, existing subcellular heating platforms capable of combining spatially confined heating, quantitative thermometry and simultaneous imaging of cellular responses remain limited. We developed a quantitative nanoheater-thermometer (qNanoHT), a polymeric nanoparticle integrating a temperature-sensitive fluorescent, dye and a photothermal dye. qNanoHT determines local temperature from fluorescence lifetime using fluorescence lifetime imaging microscopy (FLIM), thereby reducing susceptibility to photobleaching, focal drift and variations in probe concentration compared with intensity-based methods. The platform enabled real-time measurement at a subcellular heat spot while the dynamics of heat shock factor 1 (HSF1) were monitored in living cells. Heating at a single intracellular site was sufficient to induce HSF1 foci. Foci induced by mild heating at approximately 38 {degrees}C dissolved after heating ceased, whereas those induced by stronger heating at approximately 41 {degrees}C persisted and were associated with caspase-3/7 activation and apoptosis. Notably, qNanoHT-mediated subcellular heating induced HSF1 foci at a lower measured temperature than uniform whole-cell heating approximately 38 {degrees}C versus 39 {degrees}C indicating that the spatial extent of heating influences the HSF1 activation threshold. qNanoHT therefore provides a quantitative platform for relating local intracellular temperature to cellular stress responses and subsequent cell fate.

Mechanotransduction plays a fundamental role in regulating immune cell function, yet how engineered virus-like nanospikes engage mechanosensitive signaling pathways to modulate innate immunity remains poorly understood. Here, we report virus-like nanotopography as a previously unrecognized regulator of Piezo1-mediated mechanotransduction in macrophages using virus-like mesoporous silica nanoparticles (VLPSi) with tunable rigid nanospike lengths. We demonstrate a direct structure-activity relationship between nanospike geometry and Piezo1-dependent Ca{superscript 2}+ influx, with longer nanospikes inducing significantly greater intracellular Ca{superscript 2}+ signaling. Building on this mechanistic insight, we developed biomimetic cancer cell membrane (CM)-coated, MSA-2-loaded VLPSi nanoparticle (CM/MSA-2@VLPSi) and investigate the combination of nanospikes-activated Piezo1 with STING signaling and CM antigens presentation in macrophage immune reprogramming. The resulting biomimetic nanoparticles robustly activate the STING-TBK1-IRF3/NF-{kappa}B axis, increase IFN-{beta} and pro-inflammatory cytokine production, and promote macrophage polarization toward M1 phenotype in a spike-length-dependent manner. Collectively, the present study provides a biomimetic strategy for enhancing the M1 polarization of macrophage through the coordinated regulation of mechanical, inflammatory, and antigenic signals.

Antimicrobial resistance motivates the development of approaches capable of probing nanoparticle-bacterium interactions with nanoscale sensitivity. Here, synchrotron infrared nano-spectroscopy (SINS) is applied to investigate interactions between carbohydrate-coated silica nanoparticles and the Gram-negative model bacterium Escherichia coli at the single-cell level. Silica nanoparticles (SiO2) were coated with mannose, maltose, or trehalose to evaluate how surface carbohydrate chemistry influences their interactions with the bacterial envelope. Correlative electron microscopy revealed pronounced association of carbohydrate-SiO2 with the bacterial envelope, with features consistent with localization within the periplasmic region, whereas bare-SiO2 showed no detectable association. SINS measurements acquired directly on bacterial cells and at bacterium-nanoparticle interfaces revealed distinct, carbohydrate-dependent spectral signatures. Quantitative analysis of the amide I band used the I/I{beta} ; ratio, which describes the relative contributions of -helical and {beta}-sheet protein secondary-structure components, together with interface-dependent band-position analysis to characterize local spectral perturbations. Carbohydrate-SiO2 produced systematic changes in the I/I{beta} ; ratio, including at locations where nanoparticles were not directly observed, indicating that their effects extend beyond the sites of nanoparticle association. Comparison of measurements acquired on bacterial surfaces and at bacterium-nanoparticle interfaces further revealed that carbohydrate chemistry modulates both the magnitude and spatial extent of these spectral perturbations. Trehalose-SiO2 produced the largest interface-dependent amide I band shifts and a spectral component consistent with random-coil structures. Overall, these results demonstrate that carbohydrate surface chemistry modulates nanoscale protein conformational perturbations at the nano-bio interface and highlight SINS as a powerful approach for resolving chemically localized molecular responses at single-cell interfaces.