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

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.