Electrochemical biosensing of DNA with capture probe covalently immobilized onto glassy carbon surface

Abstract

In this study, an electrochemical DNA biosensor was developed based on the recognition of target DNA by hybridization detection with immobilized capture synthesized 21-mer single-stranded deoxyribonucleic acid (ssDNA) capture probe on a chemically modified glassy carbon electrode (GCE). The capture probe was covalently attached through free amines on the DNA bases using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosulfosuccinimide (NHS) cross-linking reaction on a carboxylate-terminated 4-aminobenzoic acid (4-ABA) monolayer modified GCE. The covalent immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on GCE surface. The aim of this work is to provide a well defined recognition interface for the detection of DNA. Square wave voltammetry (SWV) was used to monitor the hybridization reaction on the capture probe electrode. The decrease of the peak current of methylene blue (MB), an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The peak current of MB was found to increase in the following order: hybrid-modified GCE, mismatch hybrid-modified GCE, non-complementary modified GCE. There is decrease of the reduction current of MB intercalator with increasing concentration of target DNA with the capture probe. Fabrication reproducibility for 3 independently made electrode was ca. 9.7%, measured at 10 ng/μl of target DNA. The detection limit of the DNA biosensor was ca. 0.5 ng/μl for target DNA.