Deciphering the Effect of Protein on Different Types of Target Signals in SERS Detection

The matrix effect remains a significant challenge for the practical application of surface-enhanced Raman spectroscopy (SERS), with proteins being one of the most common interfering components. In this study, melamine (strong SERS signals, weak protein binding), rhodamine 6G (R6G; strong SERS signals, strong protein binding), and phenobarbital (weak SERS signals, strong protein binding) were employed as representative target molecules of different types. α-lactalbumin (α-La), β-lactoglobulin (β-Lg), and bovine serum albumin (BSA) were selected as the model proteins. The effects of the protein concentration on SERS performance were systematically evaluated in terms of gold nanoparticle stability and adsorption behavior. At higher protein concentrations, the SERS signals of all three analytes were significantly attenuated with signal suppression intensifying as protein levels increased. This was attributed to the formation of a complete protein corona on the gold nanoparticle surface, which inhibited particle aggregation and reduced the adsorption capacity of analyte. In contrast, at lower protein concentrations, protein-induced aggregation negatively affected the SERS signals of melamine and R6G but enhanced the signal of phenobarbital, which lacks the intrinsic ability to induce gold nanoparticles aggregation. Additionally, the interaction between phenobarbital and proteins facilitated its adsorption onto the SERS substrate, further boosting its weak signal. These findings offer insights into the impact of proteins on SERS signals of different types of targets and provide a new idea for achieving SERS detection of targets such as phenobarbital, which exhibit inherently weak signals due to their inability to induce nanoparticle aggregation on their own, yet possess strong binding affinity with proteins.