Rapid antibody-free fluorescence quenching-based protein detection using molecularly imprinted sensors

A dual-function, fluorescence quenching-based chemical sensing platform for rapid protein detection is developed using hydrogel molecularly imprinted polymer (MIP) microparticles that serve as both synthetic recognition elements and direct optical transducers. MIPs were imprinted against bovine hemoglobin (BHb) and fluorescently labelled via incorporation of fluorescein O-methacrylate, enabling direct optical transduction upon target binding. Using bovine hemoglobin as a model target, the dual-function materials exhibited high affinity (K_D = 1.91 µM) and imprinting- and selectivity factor values (2.3 and 1.7, respectively), exceeding accepted performance thresholds. A central novelty of this work is the emphasis on operational sensor performance under dynamic environments, moving beyond static equilibrium metrics. Rheological and extensional-flow characterization revealed that the molecular imprinting process induces distinct mechanical reinforcement of the polymer network, providing enhanced resistance to deformation and a higher linearity limit compared to non-imprinted controls. The fluorescence quenching assay enabled rapid, mix-and-measure detection to BHb directly from human serum, achieving a limit of detection of 8.38 nM and a limit of quantification of 25.39 nM, with recovery rated of 97.5–100.7% and minimal analytical error. This work demonstrates a robust, and scalable fluorescence sensing strategy that offers a practical alternative to antibody-based assays for protein biomarker detection with microfluidic diagnostic platforms.