Multiple pH spectral fusion and DNA methylation analysis based on SERS technology for differentiating benign and malignant lung cancer

Lung cancer mortality remains high due to the difficulty in accurately distinguishing early-stage lung cancer (LC) from benign lung disease (BLD) via conventional diagnostic approaches, and DNA methylation serves as an ideal early epigenetic marker for LC diagnosis while lacking efficient detection technologies. Herein, we developed a multi-pH fused surface-enhanced Raman scattering (SERS) strategy for label-free DNA methylation detection, enabling preliminary discrimination potential between early-stage LC and BLD. We first identified pH-dependent methylation-associated spectral markers by investigating SERS characteristics of cytosine/5-methylcytosine and methylated/unmethylated DNA standards under acidic (pH 3), neutral (pH 7) and alkaline (pH 9) conditions, and validated the specificity and universality of these markers at cellular level (A549/BEAS-2B cells). For clinical formalin-fixed paraffin-embedded (FFPE) samples, we quantified the pH-responsive intensity variations of core methylation-associated peaks in LC and BLD DNA, and further constructed a principal component analysis-linear discriminant analysis (PCA-LDA) model to compare the diagnostic efficacy of single-pH and multi-pH fused SERS spectra. Single-pH SERS detection showed limitations with low diagnostic performance, while the multi-pH spectral fusion strategy integrated complementary methylation feature information across different pH conditions, achieving a diagnostic accuracy of 0.93 for LC-BLD discrimination in clinical FFPE samples, with excellent sensitivity and specificity. This study presents a multi-pH fused SERS method for DNA methylation-associated spectral analysis, which improves the capture of complementary spectral information compared with single-pH detection and provides a label-free exploratory approach for differentiating early-stage LC from BLD.