Label-Free Kinetic Biosensing on a 3D- Printed Microfluidic Chip Integrated with an Optical Transmittance Setup

The development of reliable and affordable point-of-care (PoC) diagnostic devices is increasingly critical in the context of recurring global epidemics and pandemics. In this study, we present an optics-based transmittance biosensing experiment for HIV diagnostics, utilizing a biotinylated probe that binds specifically to a neutravidin-functionalized sensing surface. The sensing substrate consists of a thin gold-coated glass slide integrated into a custom-designed, 3D-printed microfluidic chip, which enables improved sample handling, automation, and reduced risk of cross-contamination. To ensure stable transmittance measurements, flow rate optimization was performed across a range of 9.5 to 12 mL/min, with 10.5 mL/min yielding the most stable signal and subsequently used for all experiments. The transmittance kinetics quantifying changes in optical transmission upon analyte binding were extracted from the real-time transmitogram data. These parameters were evaluated across varying probe concentrations (diluted in PBS) to establish a quantitative relationship suitable for diagnostic purposes. Furthermore, Monte Carlo simulations were employed to statistically analyze the variability and robustness of the kinetic parameters, contributing to the development of a novel diagnostic standard based on transmittance kinetics. The originality of this work lies in its integration of real-time optical sensing with microfluidics and kinetic modeling, advancing transmittance-based diagnostics as a viable platform for PoC HIV detection.