A laser-induced graphene-based electrochemical immunosensor for nucleic acid methylation detection

The detection of methylation in DNA and RNA is essential for the diagnosis and treatment of a wide range of diseases. A one-step fabricated laser-induced graphene (LIG) electrode has received increasing attention due to its good electrical conductivity, large specific surface area, ease of miniaturization, low cost and flexibility. Herein, a potential biosensor for N6-methyladenosine (m6A-RNA) and 5-methylcystosine-single strand DNA (5mC-ssDNA) detection was designed. The aim of this paper is to address the problem of detecting the m6A-RNA and 5mC-ssDNA content in cells. By stepwise modification of gold nanoparticles (AuNPs), sulfhydryl-modified nucleic acid chains, biotin-modified antibodies, and streptavidin-modified horseradish peroxidase (SA-HRP) at the LIG electrode, the peak current responses exhibited an increase proportional to the concentration of m6A-RNA and 5mC-ssDNA in the hydrogen peroxide-hydroquinone (H₂O₂-HQ) system. This method demonstrated a low detection limit of 2.81 pM for m6A-RNA and 9.53 pM for 5mC-ssDNA, with a linear detection range of 0.01 nM to 10 nM for both targets. The regression equation was determined as ΔI = 4.83 log c + 12.32 (R² = 0.9980) for m6A-RNA and ΔI = 9.82 log c + 22.09 (R² = 0.9903) for 5mC-ssDNA. Our method has good selectivity toward different detection targets of nucleic acid chains, stability for long-term storage and consecutive scanning (RSD of 9.42% and 2.08%, respectively) and reproducibility of 5 electrodes (RSD of 6.85%). This method utilizes gold-sulfur bonding to immobilize the detection target, which improves the conductivity of the LIG electrode and introduces an amplified portion of the signal by taking advantage of antigen-antibody specific binding. Thus, dual detection of m6A-RNA and 5mC-ssDNA was realized. Importantly, this approach is successfully applied for the detection of targets in spiked samples extracted from HeLa cells, suggesting its potential for clinical applications and providing a new perspective for the development of point-of care testing (POCT) techniques.