On the carbodiimide conjugation of DNA to mercapto-undecanoic acid-terminated SAMs on gold: an electrochemical and fluorescence imaging study

Bioconjugation of DNA to alkylthiol self-assembled monolayer (SAM)-modified gold surfaces via carbodiimide chemistry was studied using fluorophore labeled DNA. The extent and distribution of modification of the mercaptoundecanoic acid (MUA) SAM on a single-crystal gold bead electrode was characterized using in situ electrochemical fluorescence imaging. The uniformity of bioconjugation and the influence of the underlying surface crystallography were measured. The conditions for bioconjugation of dilute solution (1 μmol/L) of an amine and fluorophore labelled DNA (NH2-DNA-TYE665) to the N-hydroxysuccinimide (NHS)-ester activated MUA SAM were found to be successful using high concentrations of NHS and 1-ethyl-3-(3-dimethylamino propyl carbodiimide). The resulting DNA surface coverage was dependent on the underlying surface crystallography with larger coverages measured from surfaces composed of higher index planes than the atomically smoother low index planes. Some control over the DNA surface coverage was achieved by manipulating the labeling conditions, but reproducible surface modifications were challenging to achieve. The reproducibility of surface modification was shown to be a result of variance in the bioconjugation process rather than the quality of the electrode surfaces used or the initial MUA SAM as determined by capacitance. The presence of nonspecifically adsorbed DNA was observed using fluorescence imaging during electrochemistry, which made quantitative optimization difficult. Average measurements of the interfacial capacitance or the fluorescence intensity did not reveal the presence of this nonideal adsorbed DNA highlighting the need for more comprehensive surface analysis of bioconjugated electrode surfaces when prepared with carboiimide chemistry.