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Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1cc75dcorg.highwire.dtl.DTLVardef@4df8e2org.highwire.dtl.DTLVardef@10ed113org.highwire.dtl.DTLVardef@1c05bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Curcumin-functionalized gold nanoclusters are promising platforms for catalysis and drug delivery, yet the molecular determinants of their stability, morphology, and solvent response remain unclear. Here, microsecond all-atom molecular dynamics simulations are employed to investigate a 2 nm gold nanoparticle noncovalently coated with different curcumin forms, including neutral enol and trans-keto tautomers, the deprotonated enolate, and their mixtures in water-ethanol and water-methanol solvents. Layer-resolved analyses of radius of gyration, density profiles, and surface coverage reveal that neutral enol and trans forms generate compact assemblies with near-complete surface coverage, whereas enolate-rich systems adopt more expanded conformations with solvent-exposed molecules. Mixed systems preserve these intrinsic packing characteristics while improving overall coverage. Solvent substitution from ethanol to methanol reduces {pi}-{pi} stacking, strengthens Au-curcumin interactions, and increases surface coverage, yielding more compact nanostructures. Free energy and potential of mean force calculations indicate that deprotonated curcumin most effectively screens Au-Au interactions and stabilizes dispersed nanoparticles, while neutral tautomers provide moderate stabilization. Curcumin also enhances the loading of anticancer drug doxorubicin (DOX) onto Au nanoparticles, improving biocompatibility. Enolate(An)-containing systems produce extended structures with weaker membrane interactions, whereas neutral curcumin complexes form compact, positively charged assemblies that strongly bind to negatively charged cancer cell membranes. These findings clarify how tautomeric state and solvent environment cooperatively govern interfacial organization and colloidal stability, establish design guidelines for curcumin-based gold nanocarriers in catalysis, sensing, and drug delivery applications.

Self-assembled peptide-based nanostructures have diverse applications in the pharmaceutical and materials fields, but accurately predicting their self-assembly behavior without time-intensive organic synthesis and characterization remains a significant challenge. Here, we assess the effectiveness of AlphaFold3 (AF3), a deep learning model for protein structure prediction, in modeling peptide-based nanostructures and the interactions driving supramolecular self-assembly. We designed amphiphilic peptides composed of alternating hydrophobic residues (valine, leucine, isoleucine, phenylalanine) and hydrophilic residues (glutamic acid), varying both sequence length and residue order. Using AF3s multimer mode, we modeled assemblies with copy numbers ranging from 10 to 1000, generating diverse morphologies such as micelles and nanotubes. We qualitatively analyzed hydrophobic regions, secondary structures, and intermolecular interactions, while also calculating radii of gyration, packing scores, and aspect ratios using PyRosetta. Our results indicate that AF3 predicts morphologies consistent with hydrophobic driving forces and steric constraints. Increased hydrophobicity correlates with smaller radii of gyration, while higher copy numbers correspond to smaller aspect ratios (more compact structures). Longer hydrophobic segments lead to disordered structures, whereas longer hydrophilic segments promote organization. While AF3 captures systemic trends consistent with biophysical principles, comparisons to literature reveal discrepancies driven by charge effects and secondary structure bias, including an overemphasis on helical propensity (e.g., alanine-rich sequences) and sensitivity to terminal charge repulsion. Additionally, since AF3 is predisposed to predict a single assembled entity rather than higher-order assemblies such as multiple micelles or fibers, finding the optimal copy number for the best prediction requires system-specific iteration. These limitations highlight the need for complementary approaches with controlled chemical potential and environmental conditions, though qualitative agreement with experimental trends in morphology and compactness supports AF3s utility for initial structure generation. Our findings highlight AF3s potential as a user-friendly design tool for structure generation in peptide design, aiding the efficient development of functional self-assembled peptide nanomaterials.

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

Sustainable synthesis of photoluminescent nanomaterials with tuneable surface chemistry and defined biological activity remains a central challenge in green nanoscience. Here we show that the energy-input route used to carbonise a single bearberry (Arctostaphylos uva-ursi) extract precursor system exerts a decisive and mechanistically coherent influence over the surface chemistry, optical performance, and bioactivity of the resulting carbon quantum dots (CQDs). Hydrothermal processing (160 {degrees}C, 6 h) yields particles of 7.13 nm hydrodynamic diameter enriched in surface hydroxyl and carbonyl groups, a higher graphitic sp{superscript 2} carbon fraction (43.06%), and potent DPPH radical scavenging activity. In contrast, microwave-assisted synthesis yields 9.65 nm particles with a higher surface carboxylate content (O-C=O: 19.06%), enhanced fluorescence quantum yield, and increased intracellular uptake. Uptake is statistically significant in retinal epithelial cells at 200 {micro}g/mL (p < 0.001) and shows concentration-dependent accumulation in zebrafish larvae from 100 {micro}g/mL (p < 0.05). Combined XPS C 1s deconvolution and FTIR difference spectroscopy indicate that incomplete decarboxylation under microwave conditions underlies these distinct properties. Both formulations maintained full cytocompatibility across 10-250 {micro}g/mL in both RPE-1 and HeLa cells, with no statistically significant reduction in viability at any tested concentration. These findings define a synthesis-route-encoded structure property relationship that enables rational selection between antioxidant-optimised and imaging-optimised CQD formulations from an identical green precursor system.

The cardiac autonomic nervous system is a key driver of various cardiac disorders and arrhythmias. However, investigating neuronal regulation of the human heart has proven difficult due to immitted and reliable experimental models. Here, we present a novel microphysiological system utilizing a compartmentalized microfluidic device (MFD) to integrate co-cultured human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). MFD is composed of two wide-open chambers separated by microfluidic microchannels. hiPSC-SNs were characterized by confocal imaging and RT-qPCR for the expression of peripherin, tyrosine hydroxylase, and {beta}-tubulin III, as well as high levels of dopamine {beta}-hydroxylase and nicotinic acetylcholine receptors. Furthermore, patch-clamp techniques confirmed their functional maturity, showing spontaneous action potentials and positive responses to nicotine (1{micro}M). Co-culturing hiPSC-CMs and hiPSC-SNs within the MFD facilitated axonal projection into the cardiomyocyte chamber, establishing a physical connection between the two cell types. After 10 days of co-culture, functional integration was confirmed by a significant increase in the action potential frequency and beating rate of hiPSC-CMs, as recorded by patch-clamp and video motion tracking, respectively. Notably, nicotine application in the neuronal chamber accelerated these rates in hiPSC-CMs chamber, whereas the administration of the {beta}-blocker, propranolol (5{micro}M), effectively decreased the beating rates. Collectively, these data demonstrate the feasibility of differentiating hiPSCs into functional sympathetic neurons and establishing a robust neuro-cardiac interface. This microphysiological system represents a powerful platform for investigating disorders characterized by impaired neuro-cardiac interactions, offering a valuable tool for both disease modeling and pharmacological screening.

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [&le;] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [&ge;] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [&le;] 4 {micro}g mL-1, SI [&ge;] 128), A. baumannii (MIC [&le;] 4 {micro}g mL-1, SI [&ge;] 128), and MRSA (MIC [&le;] 4 {micro}g mL-1, SI [&ge;] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) centers are promising quantum sensors for intracellular measurements, yet nuclear applications remained out of reach because optically detected magnetic resonance (ODMR) signals are weak and capillary delivery is inefficient. This study addresses both constraints by optimizing the electron irradiation dose to balance NV creation and charge-state stability, and by grafting hyperbranched polyglycerol with terminal carboxyl groups (HPGCOOH) to suppress aggregation and prevent needle clogging. The optimized dose yields strong ODMR contrast while preserving fluorescence suitable for microscopy. HPGCOOH surfaces enable smooth and reproducible microinjection through fine capillaries. Using this strategy, the microinjection of ODMR-active FNDs into the nuclei of living COS7 cells is achieved, and clear intranuclear spectra comparable to cytoplasmic readouts are obtained. Furthermore, field-of-view temperature sensing across multiple cell nuclei is demonstrated, enabling quantitative and spatially resolved thermal mapping within the genomic environment. This methodology provides a practical route to nuclear quantum sensing and opens opportunities for nanoscale physicochemical measurements within the genomic environment.

A major limitation across nanoparticle delivery platforms is sequestration within endosomal compartments, which restricts access to intracellular targets despite efficient cellular uptake. Here, we show that peptide architecture can be used to control intracellular trafficking and reduce endosomal accumulation in lipid-protein nanocarriers. Specifically, we fuse R6W3 (RRWWRRWRR), an amphipathic cell penetrating peptide, to the N- or C- terminus of the nanodisc scaffold proteins and systematically evaluate its impact on membrane interactions and cellular behavior. Structural and biophysical characterization confirms that R6W3 incorporation preserves nanodisc assembly and protein-lipid interactions, enabling direct attribution of functional differences to peptide-driven interfacial effects. R6W3-functionalized nanodiscs exhibit enhanced binding and cellular uptake, with N-terminal fusion producing the strongest interfacial interactions. In live cells, R6W3-functionalization increases endocytic activity, evidenced by increased formation of clathrin-coated pits and intracellular colocalization with clathrin-coated vesicles. Notably, R6W3-funtionalized nanodiscs display reduced accumulation in early endosomes relative to unmodified nanodiscs, indicating decreased endosomal sequestration following endosomal uptake. These trafficking differences translate to functional outcomes, as doxorubicin-loaded, R6W3-functionalized nanodiscs achieve greater cytotoxicity than unmodified controls at equivalent concentrations. Together, these results establish peptide architecture as a design parameter for controlling intracellular trafficking and overcoming endosomal bottlenecks, providing a broadly applicable strategy for improving nanocarrier- based delivery systems.

AO_SCPLOWBSTRACTC_SCPLOWSpatially resolved characterization of nanomaterial (NM) distribution within cellular ultrastructure is essential for understanding NM fate and activity in biological systems. Volume electron microscopy (vEM) is uniquely positioned to address this challenge, yet fully documented quantitative pipelines that simultaneously segment NMs and cellular structures remain scarce. Here, an end-to-end analytical pipeline is presented based on the example of serial block-face scanning electron microscopy (SBF-SEM) data of tumor spheroids containing nanoparticles (NPs). A hybrid segmentation strategy is adopted: a fine-tuned Cellpose-SAM model for cells and nuclei, and an empirical Bayes approach for AuNPs. The fine-tuned model outperforms both the pre-trained baseline and benchmark experiments in Amira, and shows good generalization to 2D EM datasets of varying sample types, suggesting potential as a general-purpose segmentation model for electron microscopy. Full 3D reconstruction of NP distributions reveals preferential clustering in the perinuclear region, with a median nucleus-to-NP distance of 2.57 {micro}m and NM uptake spanning several orders of magnitude across cells. Furthermore, morphological analysis of segmented cells and nuclei using 3D shape descriptors and local curvature metrics provides quantitative access to features inaccessible from single sections. Together, these results establish a reproducible, open framework for the joint quantitative analysis of NM distribution and cellular morphology in vEM data.

Virus-like particles represent an emerging and promising vaccine platform. However, these particles are inherently mechanically soft and have limited control over particle surface architecture, thereby constraining their immunological control. Herein, we report the rational design of bioinspired virus-like porous silica (VLPSi) nanoparticles (NPs) with tunable and mechanically rigid spike architectures that function dually as antigen delivery carriers and immune adjuvants. Using ovalbumin (OVA) as a model antigen, we systematically elucidate the spiky structure-function relationship in antigen delivery and immune response. VLPSi NPs exhibit good biocompatibility, sustained antigen release, and markedly enhanced cellular uptake and endosomal escape compared with soft spike and spherical counterparts. Mechanistic investigations combining molecular dynamics simulations and proteomic analyses reveal that rigid spike architectures reduce the energetic barrier for cellular internalization and concurrently activate dual pathways involving endosomal Toll like receptors and calcium signaling. Consequently, VLPSi with long spikes elicit significantly enhanced humoral and cellular immune responses, outperforming the particles with shorter spikes, spherical shape as well as clinically used alum adjuvant. To demonstrate translational potential, bioinspired antibacterial vaccines were produced by loading Staphylococcus aureus surface protein rEsxB. The VLPSi-based vaccine elicited robust protective immunity to achieve complete (100%) survival following lethal challenge without detectable adverse effects, whereas traditional Alum-adjuvanted formulation conferred only minimal protection, with a survival rate of 10%. Collectively, this work establishes VLPSi with tunable spikes as a mechanistically controlled platform for next generation vaccines. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/720861v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1f6c13eorg.highwire.dtl.DTLVardef@1090d07org.highwire.dtl.DTLVardef@1364926org.highwire.dtl.DTLVardef@fc68ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Delivering biomolecules into pollen tubes that deliver sperm cells for plant fertilization remains technically challenging due to thick cell walls and rapid polarized growth, hindering reproductive engineering. DNA nanotechnology offers a promising alternative over current delivery methods due to their biocompatibility, programmable design, low cytotoxicity, and stimulus-responsive properties, yet their application in plants remains underexplored. Here, we provide the first demonstration of tetrahedral DNA nanostructures (TDNs) as nanocarriers for active, endocytosis-mediated uptake into Arabidopsis pollen tubes, enabling spermidine delivery that shortens pollen tube elongation through actin reorganization and ROS modulation. TDN-treated pollen tubes grew through the Arabidopsis stigma and style, underwent capacitation, and maintained attraction to ovules in a semi-in-vivo assay, preserving reproductive fitness. Furthermore, we demonstrate that functionalization of TDNs with nuclear localization signal peptide significantly enhances nuclear localization. Collectively, these findings establish DNA nanostructures as effective nanocarriers for targeted biomolecule delivery and precise pollen tube modulation, advancing crop reproductive engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/710033v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a09c01org.highwire.dtl.DTLVardef@623943org.highwire.dtl.DTLVardef@9d954corg.highwire.dtl.DTLVardef@1b4c35b_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

A systematic optimization of throughput and operational stability in Sorting by Interfacial Tension (SIFT) is presented. Reducing droplet size and enabling a broader distribution of droplet trajectories increased the number of droplets processed per sorting element, resulting in about a four fold improvement in throughput from 30 to 125 droplets per second. Throughput was further enhanced through device parallelization, with devices incorporating two and four independent sorting regions demonstrated. These configurations distributed droplets evenly across sorting elements that exhibited comparable pH sorting thresholds, indicating similar flow conditions and drag forces within each region. Among the designs evaluated, the two-element configuration provided the optimal balance of throughput, robustness, and simplicity, achieving maximum throughputs of about 250 droplets per second. Throughput and pH sorting thresholds were preserved throughout two hours of continuous sorting. The improved platform was applied to examine the relationship between cellular glycolysis and iron homeostasis at the single-cell level for Jurkat cells, revealing a subpopulation of highly glycolytic cells with significantly elevated iron uptake, consistent with prior reports linking iron regulation and T cell metabolism. Collectively, these advances expand the scale, stability, and biological applicability of SIFT, enabling large-scale functional studies while facilitating the capture of rare and metabolically distinct cell populations.

Protein-protected copper nanoclusters are promising candidates for bioimaging and therapeutic applications. However, harsh reducing conditions required for their synthesis, compromising the proteins structural integrity and bioactivity. Here, we report a one-pot, physiologically compatible aqueous synthesis for papain-protected copper nanoclusters (Pap-CuNCs) with blue photoluminescence and excellent photostability using nicotinamide adenine dinucleotide (NADH) as a biological reducing agent. The mild conditions (ambient temperature, neutral pH) enable the simultaneous formation of a metallic Cu0 core while stabilizing the helical content of the protein. This approach introduces a physiologically redox-controlled strategy for nanocluster formation, establishing physiological redox chemistry as a governing principle for controlling nanoscale structure and protein conformational stability. Spectroscopic and microscopic studies have demonstrated the presence of crystalline nanoclusters with a protein corona that undergoes -helical stabilization as revealed by circular dichroism. Notably, atomistic simulation studies reveal preferential binding of the copper core in the proteins active site, enhancing the -helical content of papain, consistent with experimental observations. Functionally, the Pap-CuNCs possess biocompatibility and serve as an effective delivery platform for 5-fluorouracil, leading to a 50-fold decrease in IC50 for HeLa cells without causing cytotoxicity to normal cells. This establishes a generalizable framework for bio-integrated nanocluster design under biologically compatible conditions. TOC Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/719779v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1ca9292org.highwire.dtl.DTLVardef@5cd82aorg.highwire.dtl.DTLVardef@173ed2forg.highwire.dtl.DTLVardef@1d8c30c_HPS_FORMAT_FIGEXP M_FIG C_FIG NADH-mediated physiological redox synthesis of papain-stabilized copper nanoclusters with enhanced -helical stability. This bio-integrated nanoplatform facilitates drug loading and delivery, resulting in a [~]50-fold increase in cytotoxicity in cancer cells while maintaining excellent biocompatibility toward normal cells.

In vitro fertilization (IVF) is essential for many couples facing infertility, e.g. in cases of low sperm count (oligospermia), where natural fertilization is unlikely. Medical microrobotics, making use of microscopic devices designed to perform targeted tasks inside the body under imaging guidance and controlled actuation, represents a promising strategy to guide sperm cells toward the oocyte. This approach may significantly reduce the time, invasiveness, and patient burden of conventional IVF, with long-term potential for in vivo assisted reproduction. Here, we report the first successful in vitro fertilization (IVF) using magnetically functionalized spermatozoa, termed magnetotactic sperm cells (MSCs), as a step toward in vivo microrobotic guidance of sperm cells for targeted artificial insemination. We present a protocol for the preparation of MSCs for their use in IVF, resulting in samples largely free of non-functionalized sperm cells (99.69% purity). We systematically evaluate the effect of particle functionalization on sperm health, including acrosome integrity, DNA fragmentation, mitochondrial membrane potential, oxidative stress, and epithelial interactions, and observe no adverse effects. Notably, MSCs showed improved mitochondrial membrane integrity compared to the control samples after two hours of incubation. Using MSCs, we successfully performed complete IVF cycles that resulted in embryos developing to the blastocyst stage at a comparable rate as non-functionalized sperm cells of the same concentration. Lower concentrations of non-functionalized sperm cells (comparable to those remaining in the MSC sample after purification) did not result in any development of embryos to blastocysts. To facilitate manipulation and translation, we implemented automated image-based recognition, magnetic manipulation, and pre-clustering routines that increased guidance efficiency and are compatible with standard IVF workflows. Together, these results demonstrate that magnetic functionalization can be applied without compromising key sperm quality metrics and can enable directed sperm guidance for assisted oocyte fertilization. This work provides a practical framework for integrating microrobotic sperm manipulation into assisted-reproduction workflows and supports further development toward automated in vitro and eventual in vivo applications.

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

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of [~]4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on one side and a bromo-thiophene tail on the other side, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with [~]65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This is the first study, to our knowledge, that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

Osmotic pressure has been known to play essential roles in living systems from single cells to complex tissues. However, direct in-situ measurements of osmotic pressures in biosystems have remained challenging, especially in complicated heterogeneous systems in which osmotic pressure gradients could exist and induce directed forces. Bacterial biofilms -- organized communities of bacteria encased in a self-produced extracellular matrix -- are a major mode of bacterial life. It has, however, remained unexplored how the osmotic pressure is distributed in the biofilm and how this distribution contributes to biofilm growth and activity. Here, liposomal nano-sensors are developed for the in-situ mapping of osmotic pressures at an unprecedented microscale resolution in real time using Escherichia coli. biofilm as a model system that develops at the surface of a hydrogel containing the nutrients. The measurements reveal osmotic pressure gradients with a radially increasing trend from the inner regions to the outer regions of the biofilm, which is associated with biofilm formation, morphology, and metabolism. The gradients likely contribute to mechanical properties, internal stresses, and nutrient transport. The sensor readouts also show that there is an osmotic pressure difference between the biofilm and the adjacent medium, which may promote biofilm expansion through matrix swelling and bacteria growth via water and nutrient uptake from the surroundings. Our novel approach based on in-situ osmotic pressure mapping in a growing biofilm reveals a sophisticated spatial regulation of physical forces, which may inspire new models and approaches in the field of mechanobiology.

Microphysiological systems (MPS) are essential for modeling tissue barriers, yet integrating electrical readouts often requires permanently sealed microfluidic architectures that limit access to open-well (direct-access) workflows used in bioscience laboratories. To resolve this issue, we present a modular approach in which functional components are added and removed from a standard MPS core using a magnetic interface. This design preserves compatibility with established open-well protocols for seeding and downstream analysis, while microfluidic perfusion or electrical sensing capabilities are added only when needed. We demonstrate this approach with an impedance-sensing module that enables continuous impedance measurements to assess barrier function. By fitting spectra to an equivalent circuit model, we quantify junctional and non-junctional electrical contributions to barrier integrity over time, alongside conventional single-frequency TEER, and complementary permeability and imaging readouts. We apply this platform across three representative use cases, including LPS-induced disruption, shear stress-mediated strengthening, and compatibility with barrier models formed above a 3D hydrogel matrix.