Design, fabrication, and characterization of microfluidic-based interferometric porous silicon aptasensors for multiplexed sepsis biomarkers detection

• Fabricated a multilayer microfluidic chip with one inlet and three outlets • Designed to ensure uniform flow velocity across three biosensing zones • Enabled simultaneous detection of CRP, IL-6, and TNF-α via RIFTS on porous silicon • Multiplex assay showed less than 7% signal variation between solo and mixed modes • Biomarker size identified as the most influential factor in sensor response Multiplex biosensors are critical tools for disease diagnosis, as they enable the simultaneous detection of multiple biomarkers, providing a comprehensive assessment of complex conditions such as sepsis, where the involvement of multiple biomarkers and non-specific symptoms poses challenges to conventional diagnostics. This study presents the design and fabrication of a multiplexed microfluidic aptasensor using porous silicon (PSi) thin films as optical transducers in reflectometric interference Fourier transform spectroscopy (RIFTS). The platform targets three key sepsis biomarkers: tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP). Detection relies on real-time monitoring of refractive index changes in the PSi layer, induced by biomarker binding to immobilized aptamers within the nanostructure. The manipulated channels design ensured uniform flow velocity across the biosensor zones, enhancing uniform conditions for testing in three microchannels. A microcontroller-programmed motorized XYZ translation stage enabled automated, precise, and simultaneous real-time monitoring at multiple reading points. The microfluidic aptasensor platform for detecting three sepsis biomarkers demonstrated signal variations below 7% between individual and mixture modes, with not significant cross-reactivity, confirming selective and multiplex performance. This work advances the development of label-free, multiplex biosensing platforms, highlighting their potential for clinical diagnostics and broader applications in diverse fields.