Patterning of Multilayer Biointerfaces Driven by Microbubble Lithography Toward Optical Biochips

The precise and non-invasive integration of biological molecules into engineered surfaces remains a critical challenge in the development of advanced biointerfaces for biophotonics applications. Traditional fabrication techniques often rely on harsh chemical processes or photolithographic steps that risk compromising biomolecular function and lack microscale spatial selectivity. In this context, Microbubble Lithography (MBL) has established itself as an optical tweezers driven microbubble-based mask-free route toward patterning of biointerfaces in order to fabricate three-layer biointerfaces entirely in situ. This work reinforces this technology further and provides important advances in the form of quantitative understanding of the controlled patterning of fluorescent reporters on a heterostructured interface enhanced by an absorbing substrate and a cross-linker to improve interfacial stability and signal-to-noise ratios. We address important questions regarding adherence, reporter and cross-linker concentrations, while also studying the science behind the formation of such heterostructures. Thus, we explain the observed twin-peak, edge-dominated fluorescence via a phenomenological model using central thermophoretic depletion and peripheral enrichment at the three-phase contact line (TPCL), and a 3D fluorescence surface visualizing peripheral enrichment. Together, these results establish MBL as a mask-free, chemically gentle, and highly controllable route to construct an optical biochip platform to fabricate biomolecular architectures for biophotonics and biosensing applications.