Covalent linking DNA to graphene oxide and its comparison with physisorbed probes for Hg²⁺ detection.

Graphene oxide (GO) has attracted extensive research interest as a platform for DNA adsorption and biosensor development. While most researchers use simple physisorption of fluorescently labeled DNA, covalent sensors are less susceptible to non-specific probe displacement and minimize false positive results. In this work, three thymine-rich DNA probes of different lengths are modified on their 3'-end with an amino group for covalent conjugation to GO. They also each contain an internally labeled fluorophore so that Hg(2+) binding can lead to a large distance increase between the fluorophore and the GO surface for fluorescence signaling. Hg(2+)-dependent fluorescence signaling from the covalent sensors are compared with that from the non-covalent sensors in terms of sensitivity, selectivity, signaling kinetics, and continuous monitoring. The covalent sensors are much more stable and resistant to non-specific probe displacement, while still retaining high sensitivity and similar selectivity. The detection limits are 16.3 and 20.6 nM Hg(2+), respectively, for the covalent and non-covalent sensors, and detection of spiked Hg(2+) in Lake Ontario water is demonstrated.