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Here, we report a fluorescent electrospun nanofiber membrane for integration into microfluidic devices towards lung-on-a-chip applications complemented with the results of computational fluid dynamics modelling. A proposed hybrid poly(ε-caprolactone) (PCL)–collagen membrane was developed, characterized, tested, and integrated into a prototype microfluidic chip for biocompatibility studies. The resulting membrane has a thickness of approximately 10 μm, can be adjusted for appropriate porosity, and offers excellent biocompatibility for mimicry of a basement membrane to be used in lung-on-a-chip device applications. Several membrane variations were synthesized and evaluated using SEM, FTIR, AFM, and high-resolution confocal fluorescence microscopy. A sample microfluidic chip made of cyclic olefin copolymer and polydimethylsiloxane was built and integrated with the developed PCL–collagen …