Precise Methylation Detection of Tumor Suppressor Gene Promoters by Magnetic Enrichment and Nano Silver Adduct-Promoted Surface-Enhanced Raman Scattering.

Noninvasive liquid biopsies can be used for early tumor diagnosis by identifying the methylation level of the tumor suppressor genes (TSGs)-a reliable index for cancer evaluation. However, identifying trace circulating genes from specimens remains challenging. This work introduces a novel method that combines magnetic isolation and surface-enhanced Raman scattering (SERS) to concentrate and detect the methylated TSG promotors. A superparamagnetic iron oxide nanoparticle modified with streptavidin is prepared as a universal magnetic bead. Biotin-terminated probe single-strand DNA (ssDNA) is immobilized on the magnetic beads through biotin-streptavidin bioconjugation. Artificial target ssDNA fragments with various methylation levels are applied as a promoter gene model. Concentrated double-strand DNA (dsDNA) is produced by a hybridizing probe and target ssDNA on magnetic nanobeads, as well as an additional magnetic isolation process. The well-prepared DNA adduct, which consists of 3 nm cisplatin-modified Ag nanoclusters, can specifically bind with guanine-cytosine base pairs of dsDNA. Ag-nanoparticle-induced localized SERS amplified signals of 5-methylcytosine (5-mC) from the dsDNA in Raman spectra, enabling accurate methylation level measurement in mixtures of 0-1 µm methylated DNA, with a detection limit of 0.05 µm. This method shows promise for enabling the methylation level evaluation of various TSGs and promoters in early cancer liquid biopsies.
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