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Frequency-shift based surface-enhanced Raman spectroscopy (SERS) has exhibited great potential applications in bioanalytical chemistry and biomedicine in recent years. The basis and the crucial factors determining frequency shifts are, however, still unclear. Herein, we have systematically investigated how solvents, antigens, and antibodies affect the band shifts in SERS-based immunoassays. By applying the charge transfer theory together with the Stark effect and time-dependent density functional theory (TDDFT) calculation, mechanistic insights into the frequency shifts in immunoreactions is proposed and discussed in detail. Accordingly, the experimental condition is further optimized and is successfully applied for the first time to detect carbonylated proteins, promising diagnostic biomarkers for human diseases. This study provides theoretical guidance for designing SERS frequency shift-based immunoassays and paves a new avenue for further applications of the strategy in clinical diagnosis.

Frequency Shifts in Surface-Enhanced Raman Spectroscopy-Based Immunoassays: Mechanistic Insights and Application in Protein Carbonylation Detection