A main challenge for optical graphene-based biosensors detecting nucleic acid is the selection of key parameters e.g. graphenic chemical structure, nanomaterial dispersion, ionic strength, and appropriate molecular interaction mechanisms. Herein we study interactions between a fluorescein-labelled DNA (FAM-DNA) probe and target single-stranded complementary DNA (cDNA) on three graphenic species, aiming to determine the most suitable platform for nucleic acid detection. Graphene oxide (GO), carboxyl graphene (GO-COOH) and reduced graphene oxide functionalized with PEGylated amino groups (rGO-PEG-NH₂, PEG (polyethylene glycol)) were dispersed and characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The influence of ionic strength on molecular interaction with DNA was examined by fluorescence resonance energy transfer (FRET) comparing fluorescence intensity and anisotropy. Results indicated an effect of graphene functionalization, dispersion and concentration-dependent quenching, with GO and GO-COOH having the highest quenching abilities for FAM-DNA. Furthermore, GO and GO-COOH quenching was accentuated by the addition of either MgCl₂ or MgSO₄ cations. At 10 mM MgCl₂ or MgSO₄, the cDNA induced a decrease in fluorescence signal that was 2.7-fold for GO, 3.4-fold for GO-COOH and 4.1-fold for rGO-PEG-NH₂. Best results, allowing accurate target detection, were observed when selecting rGO-PEG-NH₂, MgCl₂ and fluorescence anisotropy as an advantageous combination suitable for nucleic acid detection and further rational design biosensor development.
Keywords: DNA; fluorescence resonance energy transfer (FRET); graphene; ionic strength; optical biosensor; quenching.