Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine

Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single‐ and double‐stranded DNA. The minute amount of DNA required per measurement, in the sub‐nanogram regime, removes the necessity of pre‐amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low‐cost and high‐throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. Chasing nucleobase variants: Direct, label‐free high‐throughput SERS analysis in combination with chemometrics and microfluidics enables the detection and quantification of different cytosine modifications in both single and double‐stranded DNA sequences. The minute amount of DNA required per measurement, in the sub‐nanogram regime, removes the necessity of pre‐amplification or enrichment steps.