Using Disulfide DNA to Enhance Control over DNA Self-AssembledMonolayer Surface Coverage and Reduce Impedance Signal Drift

Thiolated DNA biopolymer probes are widely used for their spontaneous interactions with gold electrodes to achieve self-assembled monolayers (SAMs) of DNA. This offers an attractive class of bio-interfaces for developing point-of-care (POC) diagnostics. However, SAMs are prone to structural instability and can be challenging to reproducibly fabricate for probes of different sizes and shapes. Among methods of studying SAMs, electrochemical impedance spectroscopy (EIS) has attracted a lot of attention for its extremely high sensitivity to surface electrostatics and its label-free operation. However, the strong interfacial sensitivity also brings about susceptibility to unstable and drifting impedance signals due to the disorganization of the SAM, which has thwarted the development of EIS analytical methods. Here, we combine EIS and chronocoulometry (CC) to investigate the formation of DNA SAMs created via different methods and demonstrate the impact on the quality of SAMs via background signal drifts and DNA density fixation. Specifically, we find that enhancing stability and suppressing background drift require maximizing the density of upright DNA probes. This understanding led us to develop a protocol in which thiolated DNA probes are delivered to gold surfaces in the form of disulfide dimers. This approach not only enhances the surface density by pairwise delivery but also results in controllable probe density, and it may also intrinsically favor probes binding in the stable upright position, thereby eliminating a key obstacle for creating DNA monolayers adsorbed onto gold surfaces.