Ultra-sensitive diagnostic platform utilizing gold nanoparticle integrated in a freestanding hydrogel matrix

Fluorescence-based analysis for protein detection has been widely adopted; however, they fall short in achieving ultra-sensitive detection because of their inherently low signal-to-noise ratio at sub-picomolar concentrations. To overcome this limitation, signal amplifying materials such as gold nanoparticles (AuNPs) have been integrated into various detection platforms to enhance fluorescence signals. However, fabrication of such AuNP integrated platforms remains complex, often requiring sophisticated fabrication steps, yet none approach the ultra-sensitive detection range of 10⁰ fg mL^-1. In this study, we enabled analysis of captured protein/antibody at 10⁰ fg mL^-1 concentrations through fluorescence signals utilizing freestanding gold nanoparticle integrated freestanding hydrogel platforms. The platform is fabricated by integrating AuNPs into the hydrogel matrix using a single-step photolithographic technique. The integrated AuNPs significantly enhanced the fluorescence signals compared to controls. Notably, at fg mL^-1 levels of fluorophore, the AuNP integrated hydrogels produced a robust optical signal in contrast to negligible responses observed in controls. The platform's versatility was validated using tumor necrosis factor-alpha antibody (TNF-α Ab), achieving detection at 10⁰ fg mL^-1. By synergizing the hydrogels' porous structure with AuNPs' signal amplification, this platform successfully achieves fluorescence-based ultra-sensitive protein/antibody detection.