Multimodal Sensitivity Enhancement of Binding-based Diagnostic Assays Using Functional Hydrogels

Immunoassays are critical for clinical diagnostics, yet their performance is restricted by the physical constraints of flat surfaces used to immobilize capture molecules which limit loading capacity and induce protein denaturation. Hydrogels provide immense volumetric molecular loading capacity but translating them across diverse assay formats and readout modalities without compromising analyte permeability remains challenging. Here, we present a universal strategy for hydrogel integration into immunoassay platforms for enhanced optical and electronic biosensing. Utilizing bio-orthogonal photo-click chemistry, we developed a modular and tuneable poly(ethylene glycol) (PEG) matrix that achieves a 6,000-fold increase in molecular loading and a large mesh size for analyte diffusion. This 3D architecture demonstrates broad multimodal utility with easy integration into a range of binding-based assays enabling higher sensitivity, repeatability and multiplexability. It achieves high sensitivity enhancement for fluorescence detection in microarrays (38-fold) and microtiter plates (25-fold) and enables multiplexed readout in single wells, including from clinical serum samples. In silver metallization-based assays it boosts densitometric optical detection sensitivity (18-fold) and enables inexpensive electronic detection. In lateral flow assays, it generates 20-fold signal enhancement while enabling multiplexability and higher repeatability. By standardizing high-capacity volumetric biosensing, this scalable, plug-and-play technology empowers next generation highly sensitive, field-deployable point-of-care diagnostics.