The use of nanogels (NGs) to modulate surface-enhanced Raman scattering (SERS) activities is introduced as an innovative strategy to address certain critical issues with SERS-based immunoassays. This includes the chemical deformation of SERS nanotags, as well as their nonspecific interactions and effective "hotspots" formation. Herein, the polymeric cocoon and stimuli-responsive properties of NGs were used to encapsulate SERS nanotags containing plasmonic molybdenum trioxide quantum dots (MoO3-QDs). The pH-controlled release of the encapsulated nanotags and their subsequent localization by maleimide-functionalized magnetic nanoparticles facilitated the creation of "hotspots" regions with catalyzed SERS activities. This approach resulted in developing a biosensing platform for the ultrasensitive immunoassays of hepatitis E virus (HEV) or norovirus (NoV). The immunoassays were optimized using the corresponding virus-like particles to attain limits of detection of 6.5 and 8.2 fg/mL for HEV-LPs and NoV-LPs, respectively. The SERS-based technique achieved a signal enhancement factor of up to ∼108 due to the combined electromagnetic and chemical mechanisms of the employed dual-SERS substrate of MoO3-QDs/2D hexagonal boron nitride nanosheets. The highlight and validation of the developed SERS-based immunoassays was the detection of NoV in infected patients' fecal specimen and clinical HEV G7 subtype. Importantly, this system can be used to maintain the stability of SERS nanotags and improve their reliability in immunoassays.
Keywords: 2D substrate; SERS immunoassays; SERS nanotags; molybdenum trioxide quantum dots; nanogels; virus detection; “hotspots” engineering.