Thermal-Induced Morphological Evolution of Highly Ordered Gold Nanoarrays for Ultrasensitive Digital Biodetection

Digital biodetection has revolutionized clinical research and diagnostics by utilizing single-molecule counting to overcome the limitations of traditional analog signals. While plasmon-enhanced fluorescence (PEF) offers a rapid, enzyme-free method for signal amplification, the inconsistent distribution of chemically synthesized nanoparticles on sensing interfaces remains a major barrier to detection robustness. In this study, we address this challenge by developing highly ordered gold nanoparticle arrays using nanoimprint lithography and physical vapor deposition. We systematically investigated the effect of thermal annealing temperature on the morphological and optical evolution of these arrays. Our findings demonstrate that high-temperature thermal annealing restructures as-deposited Au nanodisks into compact, quasi-spherical nanoparticles while driving grain growth and enhanced crystalline coherence. This structural refinement blue-shifts the localized surface plasmon resonance (LSPR) by approximately 100 nm, significantly optimizing spectral coupling with Cy5 fluorophores. Arrays annealed at 750 °C yield strong dark-field scattering, excellent spectral overlap with the Cy5 emission band, and superior particle-substrate adhesion, together generating the maximum fluorescence enhancement factor. Consequently, the optimized nanoarrays achieve a one-order-of-magnitude boost in detection sensitivity, enabling an attomolar-level limit of detection (LOD) for model DNA targets in buffer. Furthermore, the platform reliably detects temporally resolved single-molecule photobleaching events even in complex biological matrices such as 1 pM target in fetal bovine serum (FBS). Overall, this work presents a robust and scalable fabrication strategy for high-performance plasmonic arrays tailored for digital bioanalysis.