Polymerase-embedded Nanocomposite Thin-film Platform for Rapid, Field-deployable Detection of Antibiotic-resistant Pasteurella multocida in the Veterinary Settings

Background: We present a polymerase-embedded nanocomposite thin-film platform for rapid, field-deployable detection of antibiotic-resistant Pasteurella multocida in veterinary settings, addressing the limitations of conventional culture-based and polymerase chain reaction-dependent methods. Aims and Objectives: The proposed system integrates covalent enzyme immobilisation, isothermal amplification and colorimetric signal transduction into a portable device, enabling sensitive and stable detection without thermal cycling. Materials and Methods: A flexible polymer matrix functionalised with carboxylate groups covalently binds Bst DNA polymerase through carbodiimide chemistry, while gold nanoparticles enhance thermal conductivity and stabilise enzyme-substrate interactions. Target detection is achieved through loop-mediated isothermal amplification of resistance genes (e.g. tetH and floR ), preadsorbed onto the film, followed by the real-time monitoring through smartphone-coupled spectrophotometry. Results: The thin film exhibits a shelf-life exceeding 6 months at 4°C, with polymerase activity retention surpassing 90% after 50 cycles. The system quantifies bacterial DNA through plasmonic shifts induced by pyrophosphate-mediated nanoparticle aggregation, eliminating the need for fluorescent labels. The platform demonstrates a limit of detection (LOD) of 50 CFU/mL, outperforming culture-based methods by the two orders of magnitude while reducing assay time from 48 h to 35 min. Conclusion: Key innovations include stabilised enzyme-polymer nanocomposites, plasmonic signal transduction and modular microfluidics tailored for high-ambient temperature environments. This work bridges molecular biology, nanomaterials and point-of-care engineering offering a practical solution for antibiotic resistance monitoring in the resource-constrained veterinary workflows.