Surface-confined FRET nanoplatform printed via pyro-EHD jet for stable and reproducible TNF-α detection

Abstract

The detection of protein biomarkers at low concentrations through fluorescence resonance energy transfer (FRET) remains challenging, as conventional homogeneous assays are often affected by uncontrolled donor–acceptor diffusion, fluorophore quenching, and limited reproducibility. In this work, we propose a two-dimensional solid-supported FRET nanoplatform enabling a surface-confined sensing strategy for the stable and reliable detection of the model biomarker Tumor Necrosis Factor-alpha (TNF-α). The approach combines a nanostructured film of fluorine-doped ZnO (F/ZnO) quantum dots deposited on a glass slide with a high-precision pyro-electrohydrodynamic jet (p-jet) printing technique. Microspots of a high-affinity peptide (P52) were printed onto the F/ZnO layer to control donor–acceptor spacing and optimize fluorophore orientation and density, ensuring efficient FRET and reduced variability between samples. The platform provides stable, reproducible, and concentration-dependent FRET signals in the ng mL⁻¹ range, with a limit of detection of 31 ng mL⁻¹, suitable for identifying elevated cytokine levels associated with inflammatory responses. Assay selectivity was evaluated in the presence of non-target proteins, including bovine serum albumin (BSA) and phosphorylated Tau (p-Tau181), and in artificial urine as a complex biological matrix. The results indicate limited interference and minimal matrix effects. Overall, this strategy offers a robust architecture with low reagent consumption and scalable fabrication for future point-of-care diagnostic applications.