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

Abstract

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.

Analytical epigenetics: single-molecule optical detection of DNA and histone modifications

“…At the same time, hybridization events could be quantified. In another study, several C modifications (among them 5mC and 5hmC) were quantified in ssDNA and dsDNA [53] (Figure 4 (b)). The authors applied positively-charged spermine-coated silver nanoparticles, which aggregate upon addition of DNA due to electrostatic interactions.…”

“…(b) Direct label-free detection of various DNA modifications by silver nanoparticles that aggregate in the presence of DNA, leading to a shift of their surface plasmon resonance and consequently a color change of the nanoparticle solution. Two SERS bands show an intensity change for the modified nucleobases 5mC and 5hmC [53]. …”

Combination of liquid‐based column separations with surface‐enhanced Raman spectroscopy

“…Obviously, the co‐adsorption might be problematic for negatively charged entities, since they are repelled from the substrate by stabilizing anions (typically citrates). In these cases, the interaction might be supported by an additive acting as a positive bridge attaching both, substrate and analyte (e.g., poly(L‐lysine) or spermine ), surface modification of substrate or pH adjustment .…”

High‐Throughput Block Optical DNA Sequence Identification

“…Since the different nucleobases in DNA are biochemically distinct, their unique interactions with light photons (observable optical fingerprints) can be used to discriminate them . Surface‐enhanced Raman spectroscopy (SERS) is an optical method routinely used for identification of unknown chemical and biochemical compounds from their vibrational fingerprints .…”

“…Applying SERS, or tip‐enhanced Raman spectroscopy (TERS), for reproducible single‐molecule DNA sequence identification has proven difficult. Previous studies have used SERS/TERS measurements on DNA for label‐free chemical fingerprinting; however, mixing of a large number of DNA molecules with metal nanoparticles provides an ensemble spectra and poses uncertainties in signal strengths . Furthermore, DNA molecules have varied enhancement due to differences in their location from the plasmonic antenna, and thus suffer from low reproducibility.…”