Cyclopamine, a typical isosteroidal alkaloid identified as a hedgehog pathway inhibitor, serves as a crucial molecular scaffold for the semi-synthesis of drugs treating nevoid basal cell carcinoma. Verazine is a key intermediate in the biosynthetic pathway of cyclopamine. In this study, we reconstructed the biosynthetic pathway of verazine in engineered Saccharomyces cerevisiae BY-SQ1, obtaining a strain that produced verazine with a yield of 265.38 μg/L. Furthermore, during a 1,000-ns classical MD trajectory, cholesterol retained a broadly consistent heme-facing pose within VcCYP90B27. The modeled pro-R C22 hydrogen remained closer to the heme Fe center on average than the pro-S hydrogen (4.01 vs 4.65 Å), revealing a persistent orientational bias in the bound ensemble. MM-GBSA and contact analyses further indicated that hydrophobic packing around the steroid scaffold and side chain may help maintain this asymmetric presentation. This research not only lays a foundation for the biosynthesis of cyclopamine and other steroidal alkaloids, but also provides a structural rationale for how cholesterol recognition by VcCYP90B27 may contribute to the observed 22R hydroxylation and identifies candidate residues for experimental evaluation.
The integration of light and cold signaling is critical for cold acclimation in plants, but the regulatory complexity of the underlying transcriptional network remains poorly understood. To systematically dissect this interplay in tomato (Solanum lycopersicum), we constructed a computational model that merges the core cold-responsive inducer of CBF expression 1 (ICE1)–C-repeat binding factor (CBF)–cold regulated factors (COR) pathway with light-sensitive modules, including the constitutive photomorphogenic 1 (COP1)–elongated hypocotyl 5 (HY5)–MYB domain protein 15 (MYB15) cascade and the phytochrome–phytochrome interacting factor 4 (PIF4)–GA-INSENSITIVE 4 (GIA4) regulatory axis. Simulations successfully reproduced the experimentally observed gene expression dynamics across different photoperiods and revealed that phytochrome activity is co-modulated by both light quality and temperature. Our model predicts that a low red/far-red ratio enhances the expression of CBF, whereas cold treatment stabilizes phyA and phyB proteins, jointly promoting cold tolerance. Additionally, the PIF4–GAI4 negative feedback loop is shown to generate sustained oscillations in SlPIF4's expression, and the cold-responsive gene SlCOR413 is predicted to exhibit low-temperature-induced oscillatory behavior. This integrative framework provides a systems-level tool for dissecting light–cold crosstalk and offers a basis for rationally engineering cold tolerance in horticultural crops through targeted modulation of transcriptional networks.
Mature adipocytes (ACs) are essential for modeling human adipose tissue physiology in vitro, yet their large size, buoyancy, and shear sensitivity make them challenging to incorporate into engineered three-dimensional (3D) constructs. This study introduces an agarose support-bath (SB) extrusion workflow for bioprinting geometrically defined adipose constructs using a gellan gum (GG) bioink. We examined the viscosity of GG solutions and SBs to identify a low-shear printability window suitable for lipid-laden cells. SB printing significantly enhanced filament stability, pore uniformity, and construct accuracy compared to free extrusion. Viability assays showed that ACs remained intact and metabolically active after 3 d post-printing, with minimal membrane damage and maintained unilocular morphology. Immunofluorescence staining revealed uniform lipid-filled cells and expression of perilipin A, while basal and stimulated glycerol release indicated retained metabolic activity. These findings demonstrate that the established SB extrusion protocol enables gentle, precise deposition of ACs using a GG-based bioink. This workflow offers a reproducible and accessible platform for generating physiologically relevant adipose cell models, supporting early-stage biological assessment, preliminary drug testing, and soft tissue engineering.
The severity of the recent Coronavirus Disease 2019 pandemic stresses the importance of analytical and biosensor research aimed at determining and curbing disease severity. It demanded an easy-to-adapt method to reflect the infectivity of viruses. The critical role of 3C-Like protease (3CLPro) in the replication cycle of coronaviruses, such as SARS-CoV-2, highlights its potential as an effective target correlated with viral activities. In this study, we aimed to develop a modular and orthogonal analytical tool for the detection and quantification of the essential protease component of coronaviruses, focusing on the enzymatic activity of the viral 3CLPro, a key component for coronavirus replication. Our approach leveraged the high sequence conservation of 3CLPro across global coronavirus strains, particularly in its substrate recognition pocket, to computationally design optimal substrate peptides. The designed sequences were orthogonal to any natural viral protein sequences. When incorporated into Gluc or FlipGFP proteins, our designs achieved modular gain-of-signal or loss-of-signal detections of 3CLPro expression levels. These antigen-focused molecular tools could facilitate the screening of effective treatments and quantitative visualization of virus-infected cells.
CRISPR/Cas technology has been transformative for genome editing, enabling the precise integration of large DNA fragments-a capability essential for advanced genome (re)writing in plants, such as for trait stacking and pathway engineering. However, several new RNA-guided systems derived from transposable elements (TEs) have shown promise for plant genome editing, thereby enriching the toolkit available for plant engineering. This review comprehensively analyzes these emerging TE-based editors, including OMEGA nucleases (TnpB, IscB, Fanzor), CRISPR-associated transposases (CASTs), and R2 retrotransposon-derived systems. We detail their distinct architectures, evolutionary origins, and unique advantages over traditional Cas9, such as their compact size, simplified guide RNAs, and staggered DNA cleavage. We critically evaluate their nascent applications in plant models, highlighting ongoing challenges in editing efficiency, delivery, and specificity. Notably, CASTs and R2 retrotransposon-derived systems show particular promise for programmable large-fragment DNA integration, offering potential solutions for next-generation plant genome writing applications. We also examine the critical link between genome editing and plant By comparing their merits and limitations, we position these systems not as replacements but as complementary tools within an expanding genome-editing toolkit. Integrating optimized TE-based editors with robust regeneration strategies is poised to unlock new frontiers in plant synthetic biology and crop improvement.
Drought and salinity are key abiotic constraints limiting the growth, development, and yield of tomato (Solanum lycopersicum). Ethylene response factors are crucial regulators that govern plants' adaptation to diverse abiotic and biotic stress stimuli. Nevertheless, the exact role of SlERF.J2 in abiotic stress resistance remains unclear. In this study, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene-editing technology was used to generate slerf.j2 knockout tomato lines to clarify the biological function of SlERF.J2 in regulating tomato's responses to drought and salt stress. The results revealed that deletion of SlERF.J2 markedly enhanced tomato seedlings' resistance to polyethylene glycol 6000-induced drought, mannitol, and salt stresses. Compared with the wild-type (WT), the knockout lines exhibited significantly increased peroxidase activity and leaf relative water content, as well as markedly decreased contents of hydrogen peroxide and malondialdehyde. After stress treatment, the expression levels of genes associated with the stress response, flavonoid biosynthesis, chlorophyll biosynthesis, light response, cell division, and hormone biosynthesis in SlERF.J2-edited tomato lines were significantly higher than those in WT plants. A yeast one-hybrid assay confirmed the direct interaction between SlERF.J2 and the SlCPS promoter. Collectively, this study clarifies the important function of SlERF.J2 in regulating drought and salt stress tolerance in tomato, providing genetic resources for tomato breeding.
Constructing expression cell lines using site-specific integration (SSI) technology has become a prominent focus in cell line development (CLD) because of its potential to minimize clonal variation. However, this technology has not yet been widely adopted in commercial biopharmaceutical manufacturing. A major reason is the low yield of SSI-derived cell lines. To improve yield, we designed landing pad locus-specific synthetic promoters by screening for potent transcription factor regulatory elements (TFREs) within an SSI platform. We then combined these TFREs to create novel, strong promoters. All synthetic promoters exhibited reporter expression comparable to that of the CMV promoter, with some up to 20% stronger, despite being only one-third the length of the CMV promoter. When expressing a monospecific antibody, fed-batch titers from these synthetic promoters were up to 45% higher than those from the CMV promoter. We also used synthetic promoters to express a bispecific antibody via SSI. The titer from a pure synthetic promoter combination was ~2.7 g/L, lower than that from an all-CMV promoter combination (~3.8 g/L). These findings demonstrate that established SSI platforms can be used to design efficient context-specific promoters, leading to significant improvements in antibody expression and greatly enhancing the value of SSI applications in CLD.
Blueberries are among the richest fruit sources of anthocyanins, which contribute significantly to fruit quality, flavor, and plant physiology. Anthocyanins also exhibit various health-promoting effects, making them valuable in the food, pharmaceutical, and cosmetic industries. Although the structures of anthocyanins and the general framework of their biosynthesis have been largely elucidated, the precise regulatory mechanisms governing anthocyanin production in blueberries remain incompletely understood. Here, we analyze recent advances in understanding blueberry anthocyanin regulation at the enzymatic pathway level (CHS, CHI, F3H, DFR, ANS) and the genetic level (MYB, bHLH, WD40 transcription factors), with particular emphasis on the roles of environmental factors such as light and temperature. This review also explores the diverse health benefits of blueberry anthocyanins, including their antioxidant properties and potential role in preventing chronic diseases such as cardiovascular disorders, neurodegeneration, and cancer. Furthermore, we discuss practical applications of anthocyanins in food, cosmetics, textiles, and pharmaceuticals, as well as emerging uses in smart packaging. Importantly, we highlight how metabolic engineering, synthetic biology, and bioprocess engineering strategies can be leveraged to enhance anthocyanin biosynthesis, stability, and yield in blueberry-based systems. By integrating insights into biosynthetic mechanisms, health impacts, and practical applications, this review provides a comprehensive framework for future research to maximize the potential of blueberry anthocyanins.
In large-scale bioprocesses, mixing limitations and design constraints cause the onset of heterogeneous environments, subjecting the cells to continuously changing external conditions, often reducing their performance compared to laboratory conditions. This study evaluated the performance in producing a heterologous transaminase (TA) of a genome-reduced Escherichia coli strain (RM214) in a STR-PFR scale-down system, benchmarking it against a wild-type strain. Under cycles of glycerol limitation and starvation, combined with oxygen limitation in later process stages, RM214 outperformed the wild-type strain. Due to its lower maintenance coefficient, RM214 showed a remarkable biomass increase of +53% and a boosted final volumetric activity with a +65% increase. These results were achieved with significantly reduced biomass-specific substrate uptake rates and respiratory parameters, both crucial for optimizing large-scale processes. This study underscores the applicability and enhanced robustness of genome-reduced strains in heterogeneous large-scale environments.
ABSTRACT Fused filament fabrication (FFF) three‐dimensional (3D) printing technologies offer new opportunities for fabricating customizable, low‐cost platforms for tissue engineering applications. Here, we developed and characterized 3D‐printed scaffolds using conductive thermoplastic polyurethane (cTPU) filaments and evaluated their mechanical, electrical, and biological performance in vitro. Dynamic mechanical analysis (DMA) across a range of temperatures and frequencies revealed that both TPU and cTPU exhibit temperature‐ and rate‐dependent elastic moduli, with cTPU showing enhanced mechanical stiffness due to the incorporation of conductive fillers. Electrical testing confirmed that cTPU exhibited a stable conductivity (∼1–2 mS/cm) resembling physiological conditions. Surface characterization showed that cTPU was significantly more hydrophilic and exhibited higher nanoscale roughness, both of which are favorable for cell‐material interactions. Mouse embryonic fibroblasts (MEFs) cultured on both scaffolds showed high viability (>85%) and significant proliferation. Notably, immunofluorescence analysis of cultured hippocampal neurons revealed significantly higher density of neuronal networks represented by higher microtubule‐associated protein 2 (MAP‐2)‐positive cell density, greater MAP‐2 area coverage, larger average MAP‐2 cell area, and enhanced postsynaptic density protein 95 (PSD‐95) expression on cTPU scaffolds. Together, these results demonstrate that FFF 3D‐printed cTPU platforms can support long‐term neuronal growth and synaptic maturation, offering promising applications in neural tissue modeling and bioelectronic interfaces. Practical Application: Characterizing soft viscoelastic materials whose properties strongly depend on temperature and strain rate is challenging and typically requires extensive testing across multiple conditions. Using a single‐specimen Dynamic Mechanical Analysis‐based mechanical testing method and a viscoelastic–elastic transformation that converts frequency‐domain viscoelastic measurements into elastic constants over a broad range of test conditions, validated by tensile tests, we efficiently generated reliable modulus data across a range of conditions, enhancing testing throughput without sacrificing accuracy. As a case study, we demonstrate the successful fabrication and comprehensive characterization of FDM 3D‐printed conductive TPU (cTPU) scaffolds for potential applications in neural tissue modeling and bioelectronic interfaces, with the results positioning cTPU composites as cost‐effective, tunable, cytocompatible, and electrically active platforms capable of supporting neuronal growth and function.
ABSTRACT Three‐dimensional (3D) printing technologies are continually advancing, significantly broadening their application scope. By employing fused deposition modeling (FDM) to create iterative prototypes and stereolithography (SLA) for crafting functional components, these additive manufacturing techniques offer a highly efficient solution regarding cost and production speed. Although biocompatible materials are widely accessible, the biocompatibility of resins remains a contentious issue and requires specific evaluation for the relevant microorganisms. This study investigates the use of commercially available resins for biotechnology applications in a lab environment. Special emphasis was placed on material reuse, ensuring compatibility with autoclaving, the most common sterilization technique in laboratories. An analytical assessment was performed to identify potential leaching of polymers or resin components into the surrounding medium during autoclaving and to examine whether the materials' mechanical properties are preserved post‐sterilization. The outcomes of this study may promote the broader use of 3D printing in biotechnology research by highlighting its sustainability, resource‐saving potential, versatility, and relevance for various laboratory applications. Practical application: 3D printing has emerged as a crucial asset in research, highlighted by its versatility and adaptability. The application of synthetic resins in biotechnological contexts using stereolithography‐based 3D printing technologies has been subject to considerable criticism in the past, primarily due to verified toxic effects. Although biocompatible resins are now commercially available, their functional performance and long‐term safety have not been sufficiently studied. This study aims to facilitate the integration of 3D printing materials into standard biotechnological laboratory workflows by examining the viability of autoclaving as a sterilization technique. Additionally, for the first time, the reusability of the materials was assessed by testing their mechanical properties after multiple uses and repeated autoclaving. This approach seeks to simplify the use of 3D printing in biotechnological research, thereby facilitating its integration into routine laboratory workflows and supporting further advancements in the field.
ABSTRACT The lipase‐catalyzed oxidative functionalization of alkenes in Pickering emulsions (PE) offers a green and efficient alternative to conventional processes involving hazardous oxidants. Other investigated reaction media used for this alternative green pathway still suffer from enzyme deactivation and /or low specific reaction rate which limits their industrial adaptability. This study investigates the enzymatic synthesis of peroxyacetic acid, the first step in lipase‐catalyzed oxidative functionalization, in a continuous membrane reactor. The effects of aqueous phase composition, modified silica nanoparticles, and operational parameters on reaction performance were systematically studied. Optimal conditions were obtained at pH 7, 100 mM buffer, and 5 g Ldp−1 enzyme concentration. Surface‐modified silica nanoparticles improved PE stability and interfacial catalytic efficiency, while maintaining comparable catalytic productivity. Hydrogen peroxide outperformed urea hydrogen peroxide, yielding a maximum product yield of 83% at a concentration of 45 mM in the influent solution and a space‐time yield of 44.9 gPAL−1d−1 at 386 mM. At 386 mM, the specific reaction rate (17.5 mmol g−1 h−1) was over twice that of reported single organic‐phase systems. Despite the high peroxide concentrations, the enzyme displayed remarkable stability in PE due to the protective role of nanoparticles. This work provides critical insights into optimizing enzymatic oxidative functionalization in PE and their potential for sustainable industrial applications. Practical application: This study provides a foundation for the development of sustainable and efficient continuous processes for oxidative biotransformations using Pickering emulsions (PE). By enabling the enzymatic production of peracids, the first step in lipase‐catalyzed oxidative functionalization, the process addresses key limitations of previously explored green reaction systems, such as enzyme deactivation and low specific activity. The PE system enhances emulsion stability and preserves enzyme activity under high oxidant concentrations, achieving high yields and space‐time productivity in a membrane‐based continuous reactor. Surface‐modified silica nanoparticles further improve interfacial catalytic efficiency as well as emulsion stability. This approach is well‐suited for selective and eco‐friendly oxidation in the synthesis of fine chemicals, active pharmaceutical ingredients, and specialty materials. Additionally, the robustness of the system allows stable operation under harsh conditions, supporting the efficient integration of the second, chemical epoxidation step. These findings contribute to the broader implementation of continuous green chemistry technologies in industrial biocatalysis.
ABSTRACT Genetically engineered plants incorporate the use of a novel bioreactor known as molecular pharming, which has a transformative view on the pharmaceutical industry. The technique enables mass production, at a low cost, and reproducibly of a large number of different protein‐based drugs, vaccines, and industrial enzymes. This review‐based study outlines the chronological evolution of molecular pharming, investigates its essential principles and elective applications, and meticulously compares it with other methods, namely conventional biomanufacturing. We present the numerous host organisms employed, the leading‐edge genetic engineering procedure, and the sophisticated approaches for protein purification and extraction. Additionally, we deliver in‐depth analysis of the noteworthy advantages that take place in molecular pharming as a captivating substitute, in conjunction with obstinate challenges, including concerns of public insights, intricate regulatory frameworks, and consideration for economic sustainability. Finally, this comprehensive study explores the promising direction, evolving innovations, and essential areas that influence future research to fully reveal the extensive potential of plant‐based biopharmaceutical production for industrial strains and global health.
Oxygen transfer is a critical design parameter in laboratory-scale bioprocess systems used for prototyping, process development, and scale-down studies of mammalian cell cultures, particularly when cultivating shear-sensitive mammalian cells. In this work, we present the design and characterization of a 3D-printed modular, membrane-based aeration module that enables bubble-free oxygen transfer in laboratory reference cell-cultivation systems. The aeration module was developed as an external, small-scale unit intended for flexible integration into laboratory bioreactors and perfusion setups. Fabricated via fused deposition modeling, the final design features a three-chamber membrane-stacking architecture that ensures mechanical stability, tightness, and biocompatibility, while allowing for straightforward adaptation through editable CAD files. The system was experimentally evaluated with respect to oxygen transfer performance under varying relative liquid flow rates and membrane configurations (PTFE and PVDF), each with two different pore sizes (0.22 µm and 0.45 µm). Key performance parameters of the aeration module were determined and include dissolved oxygen (DO) profiles, volumetric oxygen transfer coefficients (7.26 h-1), oxygen transfer rates (OTRs) (max. 61.4 mg L-1h-1), and the pressure-normalized oxygen mass transfer rate (0.87 g m- 2bar- 1h- 1). Overall, the modular design and quantified performance provide a versatile tool for rapid iteration and evaluation of membrane-based oxygenation strategies in early-stage bioprocess development.
Hesperetin is a bioactive flavonoid with potential applications in pharmaceuticals and nutraceuticals, yet its low natural abundance limits commercial use. In this study, a two-step whole-cell bioconversion process was developed for the microbial production of hesperetin from naringenin in Escherichia coli. The 4-hydroxyphenylacetate-3-hydroxylase enzyme complex (HpaBC) enabled cytochrome P450-independent conversion of naringenin to eriodictyol. Subsequent 4'-O-methylation was achieved using a plant-derived flavonoid 4'-O-methyltransferase (FOMT) coupled with a halide methyltransferase (HMT) for in situ S-adenosylmethionine (SAM) regeneration. Enzyme activity was first confirmed individually in vitro and in vivo, followed by integration into recombinant whole-cell systems, co-expressing all desired enzymes. Process optimization through delayed co-substrate addition, improving induction conditions, and machine learning-guided parameter selection increased hesperetin yields up to 70.6% with minimal byproduct formation. This work demonstrates the feasibility of combining process development and digital optimization strategies for the sustainable production of methylated flavonoids in microbial systems. The resulting E. coli platform provides a scalable blueprint for future biotechnological applications involving cofactor-dependent plant secondary metabolism.
Extrusion-based Bioprinting is a key technology in biofabrication, yet the choice of extrusion method is often limited to established techniques built into most bioprinters, limiting the print fidelity and more demanding applications like printing material gradients. In this technical report we compare the emerging method of progressive cavity pump with established technologies such as pneumatic extrusion and syringe pump-based printing setups. The three methods were compared for their accuracy and precision in extruding 35% Pluronic F127, followed by test simulating different flow profiles, material dependency and ability to transfer between hardware setups. The progressive cavity pumps showed the most advantageous behavior for gradient printing, with the syringe pumps needing more iterations for stable extrusion and the pneumatic extrusion enabling high-volume extrusion but showing lower precision. This was further shown with the printing of different gradients.
The acetogen A. woodii efficiently converts CO2 and H2 to acetate. Metabolic engineering enabled the autotrophic production of non-native products, for example, d-lactate from CO2 by overexpression of d-lactate dehydrogenase from Leuconostoc mesenteroides and knockout of the native lactate dehydrogenase. During gas fermentation with acetogens, the addition of CO leads to increased provision of reducing equivalents, and thus increased biomass formation. However, literature data reveal that already small CO partial pressures in the gas phase inhibit the autotrophic growth of A. woodii. This study aims to investigate adding 0.6%-6.0% CO to batch-operated stirred tank bioreactors with continuous gassing to study autotrophic growth and product formation with the d-lactate producing A. woodii mutant. No growth and product formation were observed with 6% CO. Surprisingly, cell growth and metabolic product concentrations are non-linearly dependent on lower CO concentrations in the inlet gas phase. Highest biomass concentrations were observed with 3% CO (3.24 g L-1, 89% improvement compared to the reference process without CO addition), and the highest d-lactate accumulation was achieved with 0.8% CO (6.2 g L-1 d-lactate, 189% improvement compared to the reference without CO) after a prolonged lag phase. In conclusion, CO-sensitive A. woodii cells need tight control of CO in syngas to affect autotrophic product selectivities.
Autologous chimeric antigen receptor (CAR)-T therapies have given hope to many cancer patients whose other lines of treatment have failed. Unfortunately, limited manufacturing capability has resulted in many patients dying while on a waitlist. Similarly, since clinical trial treatments are personalized, it is difficult to treat many patients simultaneously, resulting in longer clinical trials. Therapeutic production often takes over 4 weeks, so a product failure means that a patient may need to wait another month for treatment, putting them at severe risk for disease progression. The labor-intensive manufacturing process has led to therapeutic costs of roughly $500,000 per treatment, which can be reduced by better automation and shorter manufacturing times. The goals of this article are to review CAR-T therapeutics development, manufacturing, and treatment, and to encourage the development of data analytics-based multi-scale decision support tools for all humans "in the loop." A systems approach is needed since prior treatments and current state of health (including the immune system and microbiota), initial cell quality, manufacturing failure, bridging and lymphodepletion therapy before infusion, and supply chain management, all impact treatment success. Continuous updates as more patient data are made available can lead to better treatment recommendations and outcomes.
Biosensors are an integral part of modern medicine, are used in basic research, and are increasingly used by consumers as point-of-care and wearable devices. Meanwhile, the underlying technological approaches are rapidly expanding, including spectroscopic sensing, artificial bioreceptors, synthetic biological approaches, whole-cell biosensors, and artificial intelligence. With these diversifications in applications as well as technology, the scope and meaning of the term biosensor is blurring. This paper attempts to give an overview of the sensing approaches, with their physical, chemical, biochemical, and biological principles, and an overview of the fields of application, including nonliving systems and living systems. This leads to a comprehensive overview and a reappreciation of the term biosensor, including not only devices with a molecular biorecognition element and physico-chemical readout but also the sensing of living biological systems using physical and chemical methods, and the use of living biological systems for sensing purposes.
The COVID-19 pandemic has highlighted the need for rapid, simple, and cost-effective point-of-care testing (POCT) methods for pathogen detection. Hydrogel-based biosensing has emerged as an increasingly popular approach, offering advantages such as reagent storage and multiplexing capabilities. In this study, we have functionalized different polymer substrates to ensure an adequate adherence of methacryloyl-modified gelatin-based hydrogel spots that function as biosensors. Different hydrogel formulations were tested for their suitability in a point-of-care testchip. Our findings demonstrate the good adherence properties of amino-functionalized and subsequent methacrylated polymer materials, specifically polyethylene terephthalate (PET) and oriented polypropylene (OPP), when used as substrates for hydrogel biosensors. Moreover, we successfully identified an optimal formulation for the hydrogel ink, consisting of amino-functionalized methacryloyl-modified gelatin with a biopolymer content of 3.5% (w/w) and a photoinitiator content of 0.0875%. This formulation not only enables printing with a piezoelectric 2D printer but also exhibits excellent hydrogel stability and adherence to the functionalized substrates. These results contribute to the development of reliable and efficient POCT methods for pathogen detection, addressing the limitations of current diagnostic capabilities. The study emphasizes inkjet-based functionalization, with comprehensive characterization of printability including viscosity, surface tension, and density, and provides expanded methodological details to ensure reproducibility. Practical application: This study demonstrates the successful functionalization of PET and OPP polymers as substrates for point-of-care test chips, paving the way for advanced diagnostic solutions. By amino-functionalization and methacryloylation of the surfaces, covalent bonding with biosensors was achieved, ensuring stability and adherence. A methacryloyl-modified gelatin-based hydrogel ink, optimized for piezoelectric printing, was identified for biosensor fabrication. The selected ink minimizes fluorophore quenching, preserving biosensor sensitivity. Among the tested materials, OPP showed superior adherence due to its non-polar characteristics. These findings enable the creation of multiplexed test chips capable of detecting multiple pathogens simultaneously, addressing a crucial gap in rapid and reliable diagnostics. Although the hydrogel-based biosensors have not yet been tested with encapsulated LAMP, this integration marks the next step toward fully functional point-of-care testing. Ultimately, this research advances the development of robust diagnostic platforms, with applications in healthcare settings for timely pathogen identification and disease management.
