Highly Selective Electrochemical Detection of 5-Formyluracil Relying on (2-Benzimidazolyl) Acetonitrile Labeling

The identification of formylpyrimidines in DNA is crucial for a better understanding of epigenetics. Although many techniques have been explored to detect their content, more accurate methods of formylpyrimidine determination are still required due to the relatively lower sensitivity or lack of selectivity in current methods. Herein, an electrochemical method based on the covalent bonding of the azido derivative of (2-benzimidazolyl) acetonitrile (azi-BIAN) and the aldehyde group of 5-formyluracil (5fU) was proposed for the selective detection of 5fU in the presence of 5-formylcytosine (5fC) and apyrimidinic (AP) sites. Target DNA containing 5fU was first treated with azi-BIAN and then incubated with DBCO-PEG4-Biotin to introduce a biotin group by copper-free click chemistry. Next, the sulfhydryl group was attached to the 5' end of above DNA through T4 polynucleotide kinase-catalyzed reaction. Subsequently, the labeled DNA was assembled onto the AuNPs-modified glassy carbon electrode (AuNPs/GCE) through Au-S bonds, and the streptavidin-horseradish peroxidase conjugate (SA-HRP) was further immobilized onto the surface of the above electrode by specific recognition between biotin and streptavidin. Finally, HRP catalyzed hydroquinone oxidation to benzoquinone to enhance the current signal, which was related to the amount of 5fU in nucleic acids. This method demonstrated a good linear relationship with 5fU concentrations ranging from 0.1 to 10 nM. Moreover, the level of 5fU in γ-irradiated nucleic acids was also successfully detected, indicating that the combination of molecule-depended chemical recognition and electrochemical sensing is a promising method for the selective and sensitive detection of 5fU.