Nanoscale

The first and critical reaction in magnetic hyperthermia to induce the death of cancer cells is the production of ROS (reactive oxygen species). We previously showed that it is possible to specifically deliver iron oxide magnetic nanoparticles (IONPs) in the lysosomes of cancer cells and eradicate them by targeted magnetic intra-lysosomal hyperthermia (MILH) via the application of a high frequency alternating magnetic field (AMF) without macroscopic temperature elevation. The mechanism involves a local temperature elevation at the IONPs surface which enhances the ROS production through the Fenton reaction; ROS then peroxide the proteins and lipids of the lysosomal membrane, inducing its permeabilization and leading to lysosomal enzymes release and cell death. Fe ions, critical to produce ROS in MILH, were assumed to be released by IONPs. We thus developed PEGylated multi-cores IONPs called NanoFlowers (NF@PEG) presenting or not a SiO2 shell (NF@SiO2@PEG), the later preventing the Fe3+ release from IONPs. NF@PEG released Fe ions and produced ROS production in vitro, in acidic medium mimicking lysosome upon AMF exposure, whereas NF@SiO2@PEG did not. Surprisingly, both nanoparticles increased the ROS production in cells, induced lysosome permeabilization and cell death, and slowed down the proliferation of cancer cells with the same efficacy, upon AMF application, indicating that MILH was efficient in absence of Fe3+ release from IONPs. In contrast, Ferristatin-II, an iron uptake inhibitor, prevented the ROS production and cell death in MILH induced by both IONPs, elucidating the role of endogenous iron cations responsible for the ROS production ROS in MILH to kill cancer cells.

The protein corona influences the in vivo biodistribution of ionizable lipid nanoparticles (LNPs) in nucleic acid delivery, yet its structural architecture remains poorly defined. Using cryo-transmission electron microscopy, we visualized LNP-protein interactions in their native state. We show that, unlike the discrete "fuzzy" shells observed on hard nanoparticles, LNPs displayed no peripheral protein shell. Instead, controlled incubation and competitive "dual-particle" assays, supported by molecular dynamics simulations, indicate that LNP membranes undergo localized thickening and electron-dense remodeling consistent with lipoprotein integration rather than surface adsorption. Similar features were observed in extracellular vesicles, suggesting this behavior is shared among lipid-based carriers, and proteomic analysis identified apolipoproteins as the dominant associated proteins. Together, these findings support a model in which the biological identity of LNPs arises through membrane remodeling rather than shell-like adsorption, and provide a framework for the rational design of targeted nanomedicines. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/695162v2_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@5275d3org.highwire.dtl.DTLVardef@1b59ae4org.highwire.dtl.DTLVardef@1cc290eorg.highwire.dtl.DTLVardef@9b7bfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, ~100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.

Chronic infections persist in large part thanks to protection that biofilms afford their bacterial creators. The extracellular polymeric substance of biofilms is a hydrated matrix of DNA, polysaccharides, and structural proteins, amongst other components, through which nutrients, signalling molecules, and antimicrobial agents must diffuse to reach the bacteria within. Quantitative measurement of transport on the nanoscale within in vivo biofilms remains challenging due to optical heterogeneity, autofluorescence, active remodelling of biofilms and the ambiguity in trajectory reconstruction during single-particle tracking (SPT). Here, we present a methodological framework for measuring molecular transport in defined minimal extracellular matrix models using quantum dots as fluorescent nanoscale probes imaged with high-speed SlimVar microscopy. To establish conditions in which high-diffusivity particle trajectories can be reliably reconstructed, upper limits to quantum dot concentrations were estimated from Brownian motion. The 99th-percentile inter-frame jump distance was estimated from the three-dimensional Brownian jump distance distribution and used to define a target average nearest neighbour distance, and therefore a per-particle volume, used for calculating a concentration which minimises the probability of trajectory collision during data acquisition. Quantum dot movement was imaged at sub-millisecond frame rates and diffusion coefficients were calculated in a 20% glycerol control and in DNA nanostar hydrogels modelling minimal extracellular matrix scaffolds assembled at 250 M and 500 M. Median diffusion coefficients decreased from 94.9 m2*s-1 in glycerol to 15.9 m2*s-1 and 8.3 m2*s-1 in the 250 M and 500 M hydrogels, respectively. More broadly, this work establishes a workflow for quantitative SPT in minimal biofilm models. Rather than attempting to reproduce the full biological complexity of native biofilms, this approach provides the basis of a modular experimental framework in which individual extracellular matrix components can be incorporated sequentially and their effects on molecular transport quantified.

The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.

Underwater adhesion research increasingly draws on bioinspired systems to uncover the molecular mechanisms that enable strong interfacial binding in aqueous environments. The biofilm adhesin Bap1 from Vibrio cholerae contains a short peptide motif, SYWFFGWHTK (CP), which exhibits exceptional adhesive performance, surpassing mussel foot protein mfp5 under comparable conditions. Despite its promise, the roles of ionic environments and aggregation behavior in governing CP adhesion remain unclear. In this study, we investigate how ion identity influences CP aggregation, film formation, and interfacial properties. Using dynamic light scattering, we identify the formation of micron-scale assemblies of aggregated molecular clusters (AAMCs), with size distributions modulated by salt type. Quartz crystal microbalance with dissipation and liquid atomic force microscopy reveal that CP film formation is both surface- and ion-dependent. On gold substrates, AAMCs preferentially adsorb and collapse into rigid, smooth nanofilms, consistent with hydrophobic-driven compaction. In contrast, silicate surfaces inhibit such collapse, yielding distinct morphologies and interfacial energetics. These findings demonstrate that surface chemistry and ionic conditions jointly regulate peptide aggregation and adhesion. This work provides mechanistic insight into hydrophobic-rich peptide systems and informs the rational design of next-generation wet adhesives, with broader implications for biomaterials and peptide-based formulations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/733527v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1bd012aorg.highwire.dtl.DTLVardef@1977892org.highwire.dtl.DTLVardef@16cf79borg.highwire.dtl.DTLVardef@f405bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Innate immune agonists are promising therapeutic agents to induce immune responses against cancer. However, these agents have been limited by toxicity associated with systemic accumulation and activity in off-target cells. In this work, a targeted nanoparticle (NP) platform to encapsulate and protect the Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly(I:C)) and promote its specific delivery to antigen presenting cells (APCs), macrophages and dendritic cells, for activation of this cell population was designed. To determine NP physiochemical properties that promote APC delivery, we developed a library of NP surface chemistries formed by electrostatic adsorption of polyanion coatings onto liposomes using layer-by-layer (LbL) assembly and screened the particles on APCs and off-target cells. Dextran sulfate was identified as a promising coating to enhance specific APC delivery. We applied these design parameters to develop a poly(I:C)-loaded NP for an APC-targeted immunotherapy. In a model of metastatic ovarian cancer, the LbL NP prolonged poly(I:C) retention in the peritoneal space--with 2-fold remaining 24-48hr after administration compared to free poly(I:C)--ultimately reducing systemic accumulation and associated toxicities. Compared to free drug, the NP reduced the increase in serum levels of TNF, IL-6, and CXCL10 by 9-, 4-, and 31-fold respectively. NP-treated mice experienced lower weight loss and recovered more quickly at a higher poly(I:C) dose, indicating a widening of the therapeutic window. The NP formulation enhanced accumulation of poly(I:C) in the tumor 2-fold and activation of the target APC population compared to free drug, and ultimately slowed tumor growth and extended survival in combination with doxorubicin chemotherapy. Overall, this work demonstrates a modular NP delivery strategy to improve the delivery, safety, and therapeutic window of a TLR3 agonist.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

Poly-L-Lysine (PLL) mediates the non-specific adhesion of cells and is commonly used in Atomic Force Microscopy (AFM) measurements, to ensure that cells remain attached to the substrate. However, it is acknowledged that adhesion affects the measured mechanical properties, in particular in the case Red Blood Cells (RBCs). This results in a wide range of Youngs modulus E reported in the literature. The present study aims at providing a systematic approach to the impact of non-specific adhesion on the rheology of RBCs. It provides a correlation between the topography profile of adherent RBCs and their rheology, from weak (cPLL = 10-3 mg/mL) to strong-adhesion (cPLL = 100 mg/mL) regimes. Using RICM and AFM, we find that there is a continuum of RBC shapes promoted by adhesion, from concave to dome-shaped, as predicted by the theory of vesicle adhesion. Their elastic properties discriminate them into two populations depending on adhesion strength, where stiffer RBCs (E {gtrsim} 100 Pa) correlate with dome-shaped cells. These findings are supported by rheology measurements of the dynamic complex shear modulus G*(f): while the storage modulus increases with cell-substrate adhesion, reflective of an increased membrane shear modulus, the loss modulus remains unchanged. Finally, further analysis inspired by membrane theory shows that different deformation modes may be triggered during indentation of either weakly or strongly adhering RBCs, illustrating the limits of the Hertz model.

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations (~500 nm periodicity; Wq ~25 nm) and smaller irregular corrugations (Ra ~19.2 nm). ATP stimulation reduced SF height and width by ~39% and ~15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

Modulation of immune cells as therapeutic tools has gained significant clinical relevance in the treatment of cancer. Among them, macrophages represent a promising immunotherapeutic platform not only because they can internalize tumor material, but also because they profoundly shape the tumor microenvironment through cytokine production, antigen presentation, metabolic regulation, and modulation of other immune and stromal populations. Lipid Nanoparticles (LNPs) have enabled RNA therapies to the bedside and are thus considered the gold standard for gene delivery. However, optimizing LNPs for RNA delivery to macrophages remains an active area of investigation. Here, we propose the surface modification of unPEGylated LNPs using the Layer-by-Layer (LbL) approach for enhanced RNA delivery to macrophages. Specifically, we show that fucoidan, a sulfated polysaccharide, when at the outermost layer in the LbL process provides two physicochemical advantages to unPEGylated LNPs: (1) stability in PBS and (2) resistance to lyophilization in the presence of cryoprotectant. Additionally, fucoidan improves macrophage targeting and RNA transfection efficiency compared to previously synthesized hyaluronan-decorated LbL LNPs. Fucoidan LbL LNPs (Fuc-LNPs) preferentially accumulated in CD11b+ macrophages when co-cultured with U87 glioblastoma cells, which was not observed for control PEGylated LNPs. Furthermore, Fuc-LNPs induced a higher transfection of mRNA in primary human macrophages when compared to PEGylated control LNPs. Using the model mRNA encoding CAR@CD19, Fuc-LNPs generated CAR macrophages which mediated CD19 cell ablation in vitro. Altogether, these findings highlight the potential of the LbL strategy to modulate the targeting properties of LNPs, improving RNA delivery to human macrophages and encouraging further studies using LbL LNPs for the generation of CAR-Macrophages in the context of solid tumors.

Auxetic metamaterials exhibit negative Poisson's ratio behaviors due to their architecture of periodically arranged unit cells. Although mechanical metamaterials are well established at the macroscale, programmable auxetic units remain scarce at the nanoscale. DNA origami offers a promising platform to bridge this gap, but design principles for dynamically deformable 3D auxetic nanostructures remain largely unexplored. Here, we develop design strategies for such 3D auxetic metastructures built from wireframe DNA origami. As a model system, we use a 3D re-entrant triangular unit composed of double-stranded DNA (dsDNA) bundle edges connected by single-stranded DNA (ssDNA) joints. Using coarse-grained molecular dynamics (MD) and umbrella-sampling free-energy simulations, we examine how edge design and joint-connection scheme govern auxetic responses and the energetics of the structural transformation. Our results show that auxetic performance and deformation energetics emerge from the coupled effects of DNA bundle rigidity and connector mechanics at the joints. This study provides mechanistic insights and design guidelines for programmable auxetic motion and energetics in 3D DNA origami metamaterials, advancing the development of stimuli-responsive nanomechanical devices.

Lipid nanoparticles (LNPs) have become a clinical standard for systemically-administered nucleic acid drugs and vaccines, but the LNP pipeline for locally-delivered LNP therapies remains much less mature. Local delivery in lung represents a particularly compelling application space for DNA-LNP therapeutics, as local gene therapies could support sustained epithelial recovery and functional restoration in various lung diseases. However, locally-delivered DNA-LNPs face multiple barriers, including the limited availability of serum components that often support conventional LNP activity, and the additional delivery barriers posed by the nucleus. We generated hybrid peptide-lipid nanoparticles (hpLNPs) for pulmonary DNA delivery by using a core-shell assembly strategy to incorporate short histone-derived peptides, selected for their DNA-transport capacity, into a clinically inspired LNP formulation. In parallel, we evaluated serum pre-coating of LNPs as a strategy to boost hpLNP activity in the serum-poor airway environment. A peptide:DNA amine/phosphate (N/P) ratio of 0.9 was identified as the highest feasible ratio to permit peptide incorporation into hpLNPs while preserving DNA encapsulation efficiency at >90%, retaining hpLNP colloidal stability, and preserving the overall pKa for LNPs. At N/P = 0.9, hpLNPs showed markedly enhanced DNA delivery in alveolar lung cells, achieving up to a 17-fold increase in transgene expression compared to peptide-free LNPs. Transgene expression levels varied depending on serum concentration, with expression peaking in the presence of 6% serum. Furthermore, serum pre-coating was necessary to enable in vivo hpLNP activity following intratracheal administration in mice, yielding robust local GFP expression. Mechanistic studies in exosome-free serum revealed that peptide incorporation in the hpLNPs enhanced transgene delivery by increasing nanoparticle interactions with serum exosome components, resulting in significant enhancements to hpLNP uptake. Together, these findings identify nanoparticle- serum interactions as a critical determinant of LNP-mediated pulmonary DNA delivery and establish peptide incorporation and serum pre-coating as promising strategies to enable robust, localized gene transfer in serum-free environments.

Low response rates to immune checkpoint inhibitors (ICIs) in solid tumors are often driven by insufficient tumor-infiltrating CD8 T cells and immunosuppressive tumor microenvironment (TME). Although interleukin-2 (IL-2) potently expands and activates CD8 T cells, its clinical use is limited by rapid clearance, dose-limiting toxicity, and regulatory T cell (Treg) stimulation. Engineered IL-2 variants have not yet achieved meaningful clinical efficacy. Here, polymer-modified mesoporous silica nanoparticles displaying dense, unmodified wild-type IL-2 on their surface (IL2-NP) are developed, conferring proteolytic stability and tumor retention. IL2-NP enables avidity-mediated CD8 T cell binding and enhances proliferation and effector function without increased Treg binding or proliferation. Intratumoral IL2-NP expands CD8 T cells, increases CD8/Treg ratios, and reprograms TME through dendritic cell activation and M1-like macrophage polarization. IL2-NP induces regression of both treated and untreated distant colorectal tumors in a CD8 T cell-dependent manner. IL2-NP synergizes with ICIs and leads to complete tumor regression and immunological memory that protect against rechallenge. Treatment is well tolerated, with strong efficacy also observed in triple-negative breast and metastatic ovarian cancer models. Overall, intratumoral IL2-NP elicits robust systemic antitumor immunity, offering a promising strategy to enhance ICIs, cancer vaccines, and adoptive T-cell therapies. Graphical abstractThis work introduces a nanoparticle platform that overcomes major shortcomings of IL-2 immunotherapy by presenting wild-type IL-2 at high density on the nanoparticle surface, thereby increasing binding avidity to effector T cells. The resulting IL-2 nanoparticles enhance cytotoxic T cell expansion, reprogram the tumor microenvironment, and augment responses to immune checkpoint blockade to achieve robust ant-tumor immune response in mouse tumor models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/738558v1_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@12f8c8corg.highwire.dtl.DTLVardef@b46b1forg.highwire.dtl.DTLVardef@e4efc5org.highwire.dtl.DTLVardef@3993e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Although microplastics and nanoplastics (MP/NP) are pervasive environmental contaminants, our understanding of cellular toxicity remains incomplete, as adverse effects are often attributed to long-term intracellular accumulation, while the spatiotemporal onset of cellular damage remains poorly defined. Here, we employ chemical-bond-selective stimulated Raman scattering (SRS) microscopy and cell models that decouple continuous exposure from intracellular retention to directly visualize clinically derived MP/NP-cell interactions. Cellular stress occurs primarily during MP/NP exposure, accompanied by alterations in lipid droplet (LD) composition. In contrast, following extracellular removal, intracellularly retained MP/NP become largely inert, with recovery of lipid metabolism and cellular functions. Lipidomics identifies arachidonic acid (AA) as a key dysregulated metabolite, and SRS imaging further reveals transient, spatially confined AA enrichment in MP/NP-proximal LDs during uptake. Importantly, phospholipid coating of MP/NP attenuates LD alterations and cytotoxicity while preserving particle internalization, establishing uptake-driven metabolic stress, rather than long-term intracellular retention, as primary source of MP/NP-induced damage.

Solid-state nanopores offer label-free, real-time single-molecule sensing. However, resolving fast biomolecular transport requires high-bandwidth data acquisition while the intrinsic high-frequency noise limits recovery of informative events. Here we present a hardware-software co-designed nanopore sensing platform that combines wafer-scale low-noise device engineering with deep learning-based signal reconstruction. A low-dielectric SU8 coating on silicon nitride nanopores reduces device capacitance to the pF range and suppresses high-frequency noise by up to 5-fold while maintaining facile, controllable and reproducible fabrication. This extends usable acquisition to 40 MHz and enables capture of fast molecular features. Coupled with a reconstruction model trained on synthetic translocation events embedded in experimentally measured noise, the platform recovers transient sublevels while preserving blockage edges and temporal fidelity. Using engineered DNA molecules carrying dumbbell-like barcodes, we resolve nanometer-scale structural spacings on sub-microsecond timescales, and experimentally quantify translocation dynamics within the sub-10 nanometer regime. Dual-channel measurement on a single nanopore device further demonstrates transferability of the platform by showing robust cross-channel signal reconstruction across distinct baseline noise levels. Our approach provides a general route for reliable recovery of fast event features and may enable more information-rich single-molecule sensing across diverse biomolecular targets.

Rotating helices have been utilized for many purposes, including the transport of solid material and fluids within man-made machines, for a little over two millennia. Here, we show that the rotation of a biological helical molecule--a DNA duplex--can move water and ions through a nanoscale pore. While the rotation-induced flow of water is generated by the steric shape of the DNA molecule, an even faster transport of cations is caused by electrostatic interactions. The rotation-induced ion flux is found to depend on the cation type, offering potential utility for ion separation. Finally, we show that the torque-driven duplex can move ions against a concentration gradient, realizing the Archimedes screw principle at the nanoscale.

Lipid nanoparticles (LNPs) are conventionally produced through mixing of lipids dissolved in ethanol against a buffer containing RNA. An alternative strategy offering improved cold-chain stability involves formulating empty LNPs (eLNPs), removing ethanol, and post hoc loading (PHL) RNA into the aqueous eLNPs. The kinetics of this approach remain unknown. Here, we employ a flowthrough small-angle X-ray scattering (SAXS) setup based on a confined impinging jets (CIJ) mixer to probe PHL kinetics. We show that RNA PHL in a scalable CIJ mixer is efficient and reproducible, and that SAXS data confirms that this process concludes within ~12 ms under favorable conditions in rapid turbulent micromixing, suggesting a diffusion-limited aggregation mechanism. Favorable conditions were identified as an acidic pH 5.5 buffer combined with turbulent CIJ mixing. In contrast, PHL performed with a neutral pH 7.4 buffer using a CIJ mixer or under laminar flow with a pH 5.5 buffer resulted in inefficient PHL.

Interleukin-6 (IL-6) is a pleiotropic cytokine whose aberrant elevation drives life-threatening conditions, including sepsis, cytokine storm, and autoimmune disorders, yet existing clinical detection methods demand centralized laboratory infrastructure and multi-hour assay times incompatible with rapid point-of-care decision-making. Here, we report an impedimetric aptasensor built on a programmable tetrahedral DNA nanostructure (TDN) interface anchored to a disposable gold screen-printed electrode (Au-SPE) for the ultrasensitive, label-free detection of IL-6. By systematically varying the number of thiolated base vertices from zero to three, we establish a clear and previously unreported structure-function relationship between multipodal anchoring geometry and charge-transfer resistance modulation: tripodal thiolation yields the most rigid, upright, and electrochemically responsive interface, producing the steepest analytical signal gain upon IL-6 binding at the apex-localised aptamer. Under optimised conditions (pH 7.0, 0.05 uM TDN, MCH passivation), the aptasensor exhibits a linear dynamic range of 0.0001-0.001 pg/mL, a limit of detection of 55 ag/mL, and a sensitivity of 1.55x107 ohm (pg mL-1)-1. Selectivity evaluation against seven physiologically relevant interferents such as TNF-, BSA, glucose, urea, ascorbic acid, glycine, and cysteine confirms negligible cross-reactivity, with relative responses ranging from 0.57% to 14.35% of the IL-6 signal. Spike-recovery experiments in human serum yield recoveries of 74.0-87.6% (%RSD < 4.5%), and the sensor retains functional activity for at least 21 days under refrigerated storage. This work demonstrates that thiolated vertex number is a critical and tunable design parameter for TDN-based biosensors, offering a modular, disposable platform for sub-femtogram cytokine detection with direct applicability to early sepsis diagnosis and inflammatory disease monitoring.

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.