The project continued the development of a microfluidic point‑of‑care system originally created to identify bacterial pathogens and their antibiotic resistance profiles in diabetic foot ulcers. Building on the demonstrator from the predecessor project, the team focused on integrating new amplification methods, expanding the microarray, and creating a fully cartridge‑based workflow that could be used in clinical settings. The core technical achievements include the successful operation of the cartridge at temperatures up to 95 °C, which is essential for polymerase‑chain reaction (PCR) and recombinase‑polymerase amplification (RPA). A labeling‑PCR protocol was established that allows fluorescent probes to bind directly on the cartridge‑integrated microarray, while a multiplex RPA assay was also implemented on the same platform. The hybridisation of PCR and RPA products on the microarray was demonstrated, enabling simultaneous detection of multiple bacterial species and resistance genes in a single run.

A novel cartridge layout was designed with seven independently heated wells, allowing parallel DNA amplification and reducing overall assay time. The microarray itself was enlarged to 112 spots, with reduced spot diameter and inter‑spot spacing, thereby increasing the number of detectable targets without enlarging the cartridge footprint. This “large” array was successfully integrated into the cartridge, and the entire system was read out using a new detector from Achira Labs, which provided higher sensitivity and faster data acquisition compared with the earlier readout device. Additional work focused on improving signal intensity, validating the performance of the new system against the predecessor, and extending the array’s parameter set to include additional resistance determinants. The final experiments confirmed that the integrated amplification and detection modules operate reliably under the new thermal conditions and that the system can deliver rapid, multiplexed results suitable for bedside use.

The project was carried out by five partners: BiFlow Systems GmbH in Chemnitz, which supplied the microfluidic cartridge technology; Fraunhofer IZI‑BB in Potsdam‑Golm, responsible for device development and assay optimisation; Fraunhofer ENAS in Chemnitz, which contributed expertise in microarray design and readout; Achira Labs Pvt. Ltd. in Bangalore, India, which supplied the new detection hardware; and the Manipal Academy of Higher Education, School of Life Sciences in India, which performed validation studies. The collaboration was coordinated virtually for most of the project duration, largely because the work coincided with the COVID‑19 pandemic, which limited physical access to laboratories and delayed shipments to India. Despite these constraints, the partners managed to integrate the new modules and conduct joint experiments toward the end of the project. Funding for the effort amounted to roughly 210 000 EUR, with about 100 000 EUR provided by external grants and the remainder covering personnel, consumables, and travel. The outcome of the project is a modular, cartridge‑based prototype that can be adapted to various diagnostic assays, and the partners plan to further develop the technology for other clinical applications, including a forthcoming Horizon Europe project on neurodegenerative disease diagnostics.