ACS Applied Bio Materials

FluPep is a 16 amino-acid peptide with potent anti-viral activity against influenza A virus, including the H1N1 subtype. To understand how FluPep exerts its effects, gold nanoparticles were functionalised with FluPep, since gold nanoparticles provide a very sensitive probe that can be detected optically. Our results demonstrate that nanoparticle-FluPep conjugates did not bind to influenza virus particles in vitro. However, the nanoparticle-FluPep conjugates bound to polyanions on the cell surface, and after washing cells they remained cell-associated and were able to inhibit flu virus infectivity. Nanoparticle-FluPep conjugates bound to heparin affinity columns and their binding to cells could be reduced by pre-treatment of the cells with heparinase and neuraminidase, but not with chondroitinase ABC. These data indicated that FluPep bound to heparan sulfate and sialic acid-containing glycans in the pericellular matrix and in doing so prevented viral infectivity. The importance of the interaction of FluPep with heparan sulfate (HS) was examined by extending FluPep at its C-terminus with amino acid sequences responsible for the interactions of growth factors with heparin. The resultant two new engineered peptides, termed superFluPep1 and superFluPep2, were more potent than parental FluPep. The results demonstrate that FluPep likely exerts its antiviral activity by interfering with virus-cell interactions and that enhancing the binding of such peptides to heparan sulfate is an effective strategy to increase their anti viral potency.

Chronic wounds such as diabetic foot ulcers are typically more alkaline than healing wounds, making wound pH a valuable diagnostic and prognostic marker. However, point-of-care pH monitoring remains limited by the availability of point-of-care wound pH sensing systems that offer quantitative pH determination, low toxicity, and small portable detectors. Here, we report a colorimetric pH-sensing wound dressing that enables in situ pH detection using a conventional smartphone camera. The anionic pH-sensitive dye HPTS was loaded onto cationic microparticles and embedded within a calcium-crosslinked alginate hydrogel. Across the clinically relevant range of pH 6.0-9.0, increasing pH produced a progressively more intense yellow coloration, quantified through the blue channel of smartphone-acquired RGB images. The dressing displayed a strong, rapid, and reversible signal in vitro with low dye release. In a full-thickness excisional wound model in mice, wound pH changes were detected in vivo. The combination of a pH-sensitive colorimetric hydrogel with a conventional smartphone detector offers accessible wound pH monitoring at the point-of-care.

Conducting polymers are widely used in bioelectronic interfaces because of their mixed ionic-electronic conductivity, mechanical compliance, and compatibility with biological systems. However, their electrochemically driven structural dynamics have received little attention as a mechanism for mechanical cell stimulation. Here, we show that electrochemical actuation of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) generates mechanical cues capable of activating endogenous mechanosensitive pathways in HEK293T cells. Transparent PEDOT:PSS films deposited on ITO exhibited a heterogeneous granular morphology and underwent potential-dependent microscopic deformation during electrochemical modulation. Direct optical tracking revealed displacement of the polymer boundary, with structural changes occurring preferentially in polymer-dense regions and propagating toward the film edge. When HEK293T cells were cultured directly on PEDOT:PSS, repeated electrochemical stimulation at -240 mV produced reproducible intracellular Ca2+ responses. Pharmacological inhibition with GsMTx4 attenuated the calcium response, whereas blockade of voltage-gated sodium channels with tetrodotoxin largely preserved it, supporting the involvement of mechanosensitive pathways in the cellular response. These findings identify PEDOT:PSS as an electromechanical biointerface in which electrochemical modulation can introduce a mechanical component alongside the established electrical function of the interface. This mechanical contribution should therefore be considered when interpreting cellular responses to conducting polymer- based electrical stimulation and provides a basis for engineering bioelectronic interfaces that deliberately couple electrical control with mechanotransduction.

Three-dimensional (3D) spheroid culture provides a physiologically relevant alternative to conventional two-dimensional culture, but reliable spheroid formation in microwells depends on limiting cell-substrate adhesion. Pluronic F127 is an amphiphilic triblock copolymer that forms a hydrated surface layer, reducing protein adsorption. Here, we evaluated whether this intrinsic anti-fouling property could restore an anti-adhesive surface in used microwell plates to promote spheroid formation. Using chondrogenic ATDC5 and pancreatic {beta}-cell INS-1 cells, we characterized spheroid assembly kinetics, F127 cytotoxicity, surface hydrophilicity, protein adsorption, and spheroid morphology including size and shape factor. Both cell types formed compact spheroids within 24 hours on commercial anti-adhesive microwells. F127 coating markedly reduced water contact angle and protein adsorption, confirming increased surface hydrophilicity and reduced protein fouling. In microwells stripped of their original surface coating, F127 coating amounts of approximately 0.011-0.045 mg/cm2 consistently promoted spheroid formation across both cell types. Soluble F127 concentrations were confirmed to be non-cytotoxic up to 0.625% (w/v), while even complete dissolution of the highest tested coating amount would correspond to only 0.025% (w/v) F127. This simple, reproducible, and low-cost surface-modification strategy may provide an accessible approach for re-functionalizing microwell platforms for 3D cell culture.

Parkinsons Disease (PD) is the second-most prevalent neurodegenerative disease, often characterized by neural motor dysfunction, oxidative stress, and dopamine receptor malfunction leading to improper dopamine levels in the system. DNA tetrahedron nanostructures are a promising drug delivery agent due to their biocompatibility and properties of controlled and sustained release. In this study we evaluated the potential of using TD-mediated Levodopa delivery for a MPTP induced Parkinsons Disease model in Zebrafish larvae. The induction of Parkinsonism led to morphological behaviour changes like the presence of tremors, erratic swimming behaviour, latency, and reduced locomotor activity, even elevated reactive oxygen species (ROS) and apoptosis was observed. These effects and symptoms were alleviated when the larvae were treated using TD:Levodopa conjugates, particularly at the 1:100 ratio. At the molecular level, genes like TH, DAT, SOX2, PARKIN and apoptotic genes like BCL2 and caspases showed alteration in expression in the Parkinsonism model and post treatment was induced. This highlights the potential of using DNA nanocages as a novel drug delivery agent as therapeutic strategy for Parkinsons disease. TOCDopamine loaded DNA nanocages with the capacity to overcome biological barriers for release of dopamine with neuroprotection activity in Parkinsons disease model of zebrafish. O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/742731v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@40d78forg.highwire.dtl.DTLVardef@107f59forg.highwire.dtl.DTLVardef@18ea275org.highwire.dtl.DTLVardef@14bc089_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hydrogels are widely used in sensing, delivery, and tissue engineering because their transport properties can be tuned through material design. However, while hydrogel permeability is often characterized using small molecules, many practical applications depend on the uptake and retention of much larger species, including protein conjugates and nanoparticles. Here, we systematically investigate how polyethylene glycol (PEG)-acrylate hydrogel microparticle formulation influences accumulation of signal-generating probes spanning a broad size range. We fabricated particles across a 36-condition design space varying nominal PEG-acrylate molecular weight, polymer weight percent, and UV crosslinking dose, and related formulation-dependent probe accumulation to particle swelling behavior. Increasing nominal PEG-acrylate molecular weight and decreasing polymer weight percent produced more highly swollen particles and strongly enhanced accumulation of fluorescent streptavidin conjugates, with the largest effects observed for bulky labels such as allophycocyanin and phycoerythrin. Gold nanoparticle accumulation was even more formulation-restricted, with detectable colorimetric signal observed primarily in the most permissive formulations. These findings establish design rules linking PEG hydrogel formulation to size-dependent accumulation and show that formulations suitable for small probes may be inadequate for larger reporters. More broadly, this framework may inform the design of hydrogels for particle-based assays as well as other applications where transport of macromolecules or nanoscale materials is important.

Extrusion bioprinting requires simultaneous control of material processability, rapid post-deposition stabilization, geometric fidelity, and cell compatibility. Here, we defined an experimental biofabrication window for three visible-light photocrosslinkable protein formulations based on silk fibroin (SF), gelatin methacryloyl (GelMA), and collagen methacryloyl (ColMA) using a extrusion-bioprinting workflow and ruthenium/sodium persulfate (Ru/SPS) photochemistry. Material-specific photocrosslinking conditions were first established under 430-nm irradiation and subsequently evaluated by ATR-FTIR spectroscopy, unconfined compression, short-term ARPE-19 cytocompatibility, and printing assays. Stable bulk hydrogels were obtained using 1-Ru/SPS, 45 mW, and 5 min for SF; 2.5- Ru/SPS, 45 mW, and 7 min for GelMA; and 2.5- Ru/SPS, 45 mW, and 6 min for ColMA. The selected formulations formed compliant hydrogels with initial compressive moduli of 0.60, 0.52, and 2.55 kPa for SF, GelMA, and ColMA, respectively, without significant differences among groups. Short-term XTT assays identified the unreacted Ru/SPS-containing precursor as a major biological constraint, whereas photoinitiator-free SF and GelMA maintained metabolic activity close to the culture-medium reference. GelMA and SF generated interconnected lattices with printability indices of 0.893 and 0.963, respectively. Detailed SF analysis showed high positional accuracy (96.9%) despite substantial post-deposition spreading, demonstrating that positioning accuracy and dimensional fidelity are distinct properties. Cell-laden printing was achieved with both SF and GelMA, and independent extrusion testing showed no statistically significant reduction in short-term ARPE-19 viability after SF bioprinting. Following the complete printing and photocrosslinking workflow, both formulations contained predominantly viable cells, but only SF retained the printed grid-associated cellular architecture under the tested conditions. These findings support the concept that biofabrication performance is an operational property arising from the intersection of photocrosslinking, cytocompatibility, extrusion compatibility, geometric fidelity, and post-fabrication structural stability.

Gastrointestinal infections are a leading cause of death and morbidity worldwide. Antibiotics as current treatments are ineffective or not recommended due to the emergence of multidrug resistant organisms. The initial step of the pathogenesis for most of the enteric infections is the binding of the pathogen to oligosaccharide receptors on the surface of host cells, making these receptors attractive targets for novel antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed an alternative therapeutic intervention based on a novel microbiome-based chemical engineering approach. We synthesized cholesteryl-peptides as flexible chemical scaffold for the presentation of carbohydrate GM1 mimic receptors in probiotic bacteria acting as a decoy for cholera toxin neutralization. The GM1-cholesteryl-peptides were stably embedded into the membranes of both gram positive and gram negative probiotics, Lactobacillus reuteri DSM and Escherichia coli Nissle 1917 respectively. The resulting GM1-coated probiotics demonstrated to quench nearly 100% of cholera toxin in vitro, functioning as decoys by preventing toxin binding to epithelial cells. The present study strongly suggests that our new and versatile biomimetic non-recombinant system could be employed to neutralize the functional effect of infections mediated by oligosaccharide receptors.

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.

Wet adhesives that perform under water have broad applications in industrial and biomedical settings. To date, molecular designs for underwater adhesives have largely been inspired by marine animals including mussels and barnacles. Here, we propose bacterial biofilms as an alternative source of inspiration for underwater adhesives. Specifically, we demonstrate the application potential of a peptide derived from biofilms formed by the notorious pathogen Vibrio cholerae. We characterize the ability of this biofilm-derived peptide to adsorb onto various surfaces and to glue wet surfaces, by using a combination of confocal microscopy, molecular dynamics simulations, atomic force microscopy, lap shear tests, and spectroscopic tools. We further show that the peptide can co-aggregate with microspheres acting as an effective flocculant. Finally, we succeeded in purifying this peptide from E. coli in a functional form, setting the stage for large-scale production. Our results open new design possibilities for underwater adhesives inspired by natural biofilms. TeaserA peptide derived from biofilms adheres to diverse surfaces and shows promising potential as a wet adhesive and flocculant.

Synthetic optical skin models offer reproducible, tunable optical properties but lack biological responsiveness, while tissue engineered skin models provide cellular authenticity but suffer from optical variability and limited controllability. The growing demand for alternatives to animal models in the development and validation of optical biomedical technologies highlights the need for a new class of test system that combines the strengths of both approaches while addressing their respective limitations. Here, we introduce the concept of a modular biohybrid skin model, a new testing concept that integrates an optically defined artificial epidermal layer, incorporating polydopamine nanoparticles for changes in skin tone, with living human keratinocytes in two and three-dimensional configurations. In the Optical Protection Model, UV-B-induced apoptosis in primary keratinocytes is quantitatively modulated by model pigmentation level, demonstrating a relationship between optical attenuation and caspase 3/7 activity across three artificial skin tone conditions. In a Structured Dermal Model, keratinocytes seeded onto a hydrogel scaffold localize within follicle-like microcavities, as confirmed by live/dead staining and confocal z-stack imaging. Together, these experiments lead to a new category of test system in the space between inert optical models and variable tissue models that may contribute to reducing the reliance on animal models in biomedical optics.

Gold electrodes are attractive substrates for bioelectronic and cell-free synthetic biology platforms because they are conductive, chemically stable, biocompatible, and readily functionalized through thiol-gold chemistry. Here, gene-length DNA monolayers assembled on planar gold electrodes as reusable templates for cell-free protein expression are investigated. Using thiol-modified sfGFP genes, DNA surface density is shown to be tunable by changing the DNA concentration during incubation, with the immobilized genes able to support cell-free sfGFP expression directly from the electrode surface. Further, the effects of applied voltage, storage, repeated reactions, reducing agents, and protein fouling on monolayer stability and expression output are examined. While some conditions lead to loss of reusable expression activity, dense chemisorbed monolayers can retain partial function under neutral, non-reducing conditions and are relatively robust to protein exposure. In contrast, low-density physisorbed monolayers show a stronger relationship between DNA loss and expression output. Finally, when using gold-mediated fluorescence quenching to monitor changes in DNA conformation, surface-bound DNA demonstrates electrophoretic addressability. Together, these results establish DNA- functionalized planar electrodes as a promising foundation for modular, addressable cell-free expression platforms.

Microlasers based on whispering-gallery modes (WGMs) provide bright, spectrally narrow emission, making them ideal optical probes for high-resolution cellular tagging, single-cell tracking, and biosensing. To reproducibly apply WGM microlasers in biomedical or tissue-engineering contexts, control over the number of lasers in a cell population is highly desirable. Here we use a commercial instrument for flow cytometry-based cell sorting to obtain HEK293T cell populations with a controlled number of polystyrene laser particles (LPs). By modifying the particle surface with poly(ethyleneimine) (PEI) or lipofectamine, we enhance LP-association to cells. We demonstrate that our sorting procedure yields viable cells showing unchanged proliferation rates. Manufacturer-dependent differences in particle-cell interactions were observed and investigated by scanning and transmission electron microscopy (SEM/TEM) and flow cytometry. Finally, we show that the same sorting approach enables the generation of multi-cellular spheroids with a defined ratio of LP-containing and LP-free cells. This work lays the foundation for larger tissue engineering studies with defined LP populations and control over LP-placement with respect to the cell.

Mechanical characterization of biomaterial scaffolds is essential to evaluate their capacity to meet the functional demands of target tissues in tissue engineering and regenerative medicine applications. Scaffolds designed to interface with living tissues must support the transmission of mechanical cues to resident cells and stimulate mechanosignaling pathways that are essential to their function. In joints, bone and cartilage cells act as primary mechanosensors, converting mechanical stimuli into biochemical signals that regulate tissue homeostasis and remodelling. Therefore, evaluating cellular mechanoresponses to scaffold-transmitted compression in vitro can inform the development of functional tissue-engineered constructs. For example, poly({epsilon}-caprolactone) (PCL) scaffolds are highly relevant for bone and cartilage tissue engineering due to their biocompatibility, stable mechanical properties and slow degradation. Here, we applied a custom-built device to study compression-induced mechanosignaling in MC3T3-E1 pre-osteoblast cells. The device is composed of a polydimethylsiloxane (PDMS) pillar, a force-sensing load cell, and a piezoelectric linear track. A protocol is described in which MC3T3-E1 cells are repeatedly compressed, while in parallel live tracking of force measurements and live imaging of intracellular calcium dynamics in MC3T3-E1 cells are recorded. PCL scaffolds fabricated by melt electrowriting (MEW) were subsequently integrated into the platform. Scaffold-transmitted compression triggered dynamic increases in cytosolic calcium; in MC3T3-E1 cells located directly under the PCL microfibers, but also in cells located in the interfiber spaces. This device and workflow facilitate in vitro investigations of real-time cellular mechanoresponses to dynamic compression applied with biomaterial scaffolds, and provides a testing platform for evaluating the mechanotransductive properties of scaffolds intended for tissue engineering applications.

Intranasal administration provides a noninvasive route for local and systemic drug delivery, yet its clinical effectiveness remains limited by rapid mucociliary clearance and short drug residence times within the nasal cavity. Existing stimulus-responsive nasal hydrogels seek to address this challenge but rely on physiological triggers such as temperature, pH, or ionic strength to induce gelation, resulting in variable performance due to natural differences in the nasal cavity. Here, we present a polyethylene glycol (PEG)-based hydrogel platform that overcomes these limitations through rapid in situ gel formation that is independent of physiological stimuli. The two liquid precursor solutions remain sprayable during administration and rapidly crosslink upon deposition to form a cohesive hydrogel depot. Although this dual-component design presents challenges for compatibility with conventional nasal spray devices, we demonstrate successful delivery using commercially available dual-barrel spray systems. The hydrogel maintained spray performance and regional nasal deposition comparable to conventional aqueous formulations while significantly reducing mucociliary transport to prolong mucosal residence. Importantly, reduced mucociliary transport was achieved without impairing ciliary function or compromising epithelial barrier integrity, addressing key safety considerations for repeated intranasal administration. In addition, the hydrogel altered epithelial uptake and transport of biologics, suggesting its potential to improve the nasal delivery of macromolecular therapeutics that typically exhibit poor bioavailability. The results of these study establish a versatile and clinically translatable approach for enhancing intranasal drug delivery across a broad range of therapeutic applications.

Total laryngectomy is performed for advanced laryngeal and hypopharyngeal cancers. Following the surgery, the insertion of indwelling silicone-based voice prosthesis (VP) implants is considered the gold standard for speech rehabilitation. However, early VP failure due to biofilm formation and invasion of the VP surface can lead to routine use of nystatin and the need for frequent valve changes. Despite these burdens, the composition and impact of colonising pathogens on VP integrity are poorly understood. We have developed a rapid pipeline which permitted the transfer, fixation, staining, and imaging of explanted VPs from patients within 90 minutes of sample collection. Using this pipeline, we demonstrate a multimodal reflection contrast and fluorescence confocal laser scanning microscopy technique to simultaneously map the VP surface and visualise the biofilms in situ, providing new insights into the role of pathogens on VP failure. Our results reveal that complex fungal and bacterial biofilms form polymicrobial biofilms on the VP surface. We demonstrate that biofilms not only reside on the VP surface but can invade the device, releasing silicone particles that are visible within the volume of the biofilms and are separate from the VP surface. Our findings highlight the complexity of the biofilms that form on speech valves; complex polymicrobial biofilms require more advanced treatment than what is conventionally used. Work should focus both on the patient factors that may influence this and the material properties of the valve.

Proper alignment of cells is crucial for functioning of various tissues such as skeletal muscle tissues, neural cells, adipose-derived stem cells, etc. Current in-vitro fabrication methods to replicate the cellular environment, e.g., photolithography and 3D printing, are not cost-effective and cannot capture the complexity of the surfaces to which these cells are exposed to. In this work, we used bio-mimicked leaf templates to closely resemble the in-vivo environment the muscle cells and cultured C2C12 cells, myoblast cell lines, on modified PDMS substrates fabricated using these leaf templates. Using image analysis software, we analyzed the degree of alignment of cells, aspect ratio and the area projected by individual cells cultured on these surfaces. The C2C12 cells cultured on the PDMS substrates formed utilizing the front and back sides of the leaves of Musaceae Banana were found to have an Aspect Ratio of 6.3 and 8.3, the highest among the surfaces studied in this paper. C2C12 cells cultured had the highest degree of alignment on the negative replica of the back side of Dracaena Sanderiana. Due to the availability of a wide range of leaf templates and bio-mimicked surface structures to measure cell response, it is difficult to find the optimal design. Hence, we have also tried to create a catalog using 15 different leaf surfaces and characterized these surfaces into various categories based on the grooves on the surfaces to provide a more comprehensive set of surface designs for studying cell behavior. To quantitatively analyze the groove pattern, 2D FFT analysis was also performed to find the dominant wavelength of the grooves. In surface characterization, hydrophobicity is also a parameter that needs to be considered; hence, the water contact angle of these surfaces was also measured. Our findings highlight the importance of surface topography and hydrophobicity in influencing cell alignment and can contribute to developing biomimetic surfaces for tissue engineering applications.

Despite their promise, lipid nanoparticle gene delivery systems have repeatedly failed clinical trials and struggle to achieve efficient, localized transfection in target tissues. The majority of endocytosed nanoparticles are degraded before nucleic acid release, and an inability to track particle distribution in vivo prevents validation of successful delivery. Alternatively, nanobubbles (NBs) are lipid-shelled, gas-core preclinical ultrasound contrast agents and stimuli-responsive drug delivery vehicles. Under varying acoustic pressures, NBs expand, contract, and burst, releasing cargo in an externally controlled, site-specific manner while scattering unique echoes for simultaneous ultrasound visualization. Here, we introduce a cationic nanobubble (CNB) formulation with a +42.3 mV zeta potential, 265 nm diameter, and 2.43x1011 NBs/mL concentration. CNBs produce stable ultrasound contrast, electrostatically load plasmid DNA onto their surface, and internalize into >99% of human prostate cancer cells within 15 minutes in vitro. CNBs remain brightly echogenic intracellularly and induce sonication-dependent expression of green fluorescent protein (GFP). In vivo, CNBs generate contrast in mouse livers for 50 minutes after intravenous administration. Therapeutic ultrasound stimulation over the liver causes a sharp reduction in ultrasound contrast, visualizing localized cavitation in the target organ and inducing a 2.5-fold increase in anti-GFP mean fluorescence intensity relative to the untransfected control. Importantly, no GFP expression is observed without ultrasound stimulation, supporting a mechanism for selective and site-specific gene delivery. This study presents a highly stable CNB capable of efficient DNA loading and ultrasound-dependent gene expression. These results provide a foundation for the future development of CNB platforms to advance image-guided, ultrasound-triggered gene therapy.

Two-photon polymerization (2PP) enables fabrication of hydrogel constructs with submicron, cell-scale resolution, but hydrogel-based bioinks are markedly more sensitive to process variability than conventional photoresists, and this sensitivity is further amplified when living cells are embedded in the resin. Post-processing evaluation, performed only after development, occurs too late to enable any corrective action. A full-factorial design of experiments across laser power and scan speed shows that fabrication outcome depends on both parameter choice and cell presence, with cells shifting and broadening the range of conditions yielding structurally sound constructs. However, substantial variability persists within each nominal condition and cannot be resolved by parameter refinement alone, indicating that outcome is governed by what occurs during each individual print rather than by the parameters set. To capture this, a layer-wise polymerization score is derived from pairwise comparisons of same-layer coaxial images, grounded in the psychophysics of relative judgment, and assembled into a Layer-wise Image Trajectory (LIT) for each print. Applied to both acellular and cell-laden formulations, LIT curves separate cleanly by post-processing outcome without any outcome label used in training, showing that fabrication quality can be predicted early in the build. Building on this signal, individual LIT curves are compared against statistical control limits derived from confirmed successful prints, enabling early detection of anomalous fabrication behavior at early-to-mid layers, well before development. To the best of the authors knowledge, this is the first application of in situ quality prediction and anomaly detection to cell-laden two-photon polymerization.

Rapid, selective detection of bacterial pathogens remains a central challenge. Here we report a label-free electrochemical biosensing approach that leverages protein-protein interaction (PPI)-derived peptides as recognition elements for rapid detection of Listeria monocytogenes (LM). The sensor design is inspired by the interaction between the LM virulence factor Internalin A (InlA) and the human host receptor E-cadherin (E-Cad1). Peptides derived from the InlA-binding domain of E-Cad1 were engineered as molecular recognition elements, with the E-Cad1(15-24) peptide displaying micromolar affinity and selective binding towards LM. Immobilization of these peptides on gold electrodes enabled bacterial detection by electrochemical impedance spectroscopy within 10 minutes, without labels or external signal amplification. A low peptide surface density was associated with enhanced binding-site accessibility and may facilitate multivalent interactions between the bacterial surface and the immobilized peptides. The platform produced a detectable response at experimentally tested concentrations as low as 1 CFU mL {superscript 1} and exhibited excellent selectivity under the conditions examined. This work introduces a chemically programmable, PPI-inspired biosensing paradigm that uses a reductionist approach and could potentially be extended to other pathogen targets.