Beyond the 2D Plane: Interfacing Microwave Resonators with Bioreceptor-Functionalized Hydrogel Micropillars for Label-Free Enhanced Sensitivity Biosensing

Facile and quantitative detection of liquid biopsy biomarkers such as microRNAs offers significant potential for precision healthcare; however, conventional biosensing methods rely on enzyme- or label-based workflows that are costly, time-consuming, and labor intensive. Microwave biosensors, particularly split-ring resonators (SRRs), offer an attractive alternative as they enable label-free, noncontact electromagnetic detection through permittivity measurements and are compatible with printed-circuit-board manufacturing. However, the sensitivity of conventional SRR platforms remains insufficient for clinically relevant biomarker detection. Here, we introduce an enzyme-free, label-free microwave biosensing architecture that integrates SRRs with microfluidic channels containing localized bioreceptor-functionalized hydrogel micropillars. Target hybridization within the hydrogel micropillars induces localized changes in complex permittivity, which are transduced into concentration-dependent shifts in the resonant frequency of the SRR capacitive gap. As a proof of concept, the platform is applied to detect the cancer-associated biomarker miR-16-5p using peptide nucleic acid (PNA) probes, which were selected for their neutral backbone, enzymatic stability, and strong hybridization affinity. The hydrogel micropillars act as three-dimensional scaffolds that enhance probe loading and maximize volumetric electromagnetic interaction, representing a departure from conventional planar biointerfaces. Compared with equivalent planar systems, this architecture achieves approximately a 20-fold improvement in detection limit, reaching subnanomolar sensitivity without any amplification or labeling while maintaining single-nucleotide specificity and strong device reproducibility. Beyond being the first demonstration of SRR-based miRNA detection, this work establishes a general strategy for three-dimensional microwave biosensing and positions hydrogel-interfaced resonators as a next-generation platform for sensitive, selective, label-free, and reusable biosensors.