On the Nature of DNA Self-Assembled Monolayers on Au: Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence Microscopy

Abstract

The creation of gold surfaces modified by single- or double-stranded DNA self-assembled monolayers (SAMs) is shown to produce heterogeneous surface packing densities through the use of electrochemical studies coupled with fluorescence imaging. The modified surfaces created by direct adsorption of thiolate DNA [followed by passivation with mecaptohexanol (MCH)] resulted in regions covered by a monolayer of DNA SAM and other regions that were coated by large particles of DNA. The difference in fluorescence intensity measured from these regions was dramatic. More importantly, a regional variance in fluorescence intensity in response to electrochemical potential was observed: the large aggregates showing a significantly different modulation of fluorescence intensity than the monolayer-coated regions. Electrochemical desorption and detection of the fluorescently tagged DNA provided clear evidence of a complete surface modification. These studies have implications for biosensor/biochip development using DNA SAMs. A modification in the method used to produce the DNA SAMs resulted in a significantly different surface with much fewer aggregates and more significant electromodulation of the fluorescence intensity, though at much lower DNA surface density (ca. 1% of maximum theoretical coverage). This method for forming the modified surfaces has clear advantages over the currently accepted practice and emphasizes the importance of studying the nonaveraged nature of the sensor surface using in situ imaging tools like electrofluorescence microscopy.

In Situ Fluorescence Microscopy Study of the Interfacial Inhomogeneity of DNA Mixed Self‐Assembled Monolayers at Gold Electrodes

“…the electrode was immersed first in MCH, then in DNA), much more homogeneous layers were obtained. [20] Interestingly, these results were recently confirmed at the molecular level by Ye and Josephs, [27] who performed high-resolution atomic force microscopy experiments and reached similar conclusions regarding the influence of the preparation procedure on the spatial homogeneity of DNA SAMs.…”

“…Such coupling was performed by Murphy et al, [20] who devised a unique in situ combination of fluorescence microscopy and electrochemical methods, performed in a specially designed spectroelectrochemical setup. [21,22] This technique provides spatial information, at the typical resolution of optical microscopy ( % 0.5 mm), on the homogeneous or heterogeneous nature of SAMs, and it is thus highly complementary to surface-averaged electrochemical data.…”

“…In their work, Murphy et al [20] took advantage of this phenomenon to investigate the spatial heterogeneity of DNA SAMs by monitoring fluorescence intensity variations associated with modulation of the applied potential in real time. Mixed SAMs of DNA (single and double strands) and mercaptohexanol (MCH) were prepared and studied by in situ electroMixed self-assembled monolayers composed of a fluorescently labeled DNA and a mercaptobutanol diluent immobilized on gold electrodes were characterized by electrochemical measurements coupled with in situ fluorescence microscopy.…”

Hydration Induced Stress on DNA Monolayers Grafted on Microcantilevers

“…17,18 The final strand density in self-assembled monolayers plays a critical role in nanomechanical sensing 9 and it is indeed far from being fully controllable. 19,[20][21][22][23] One singularly limiting difficulty has been to accomplish the anchoring of the ssDNA probes with a well-defined uniform density and a standing up conformation. 19,24 For such purpose, sophisticated nanografting methods need to be applied.…”

p‐Nitrobenzoic Acid Adsorption on Nanostructured Gold Surfaces Investigated by Combined Experimental and Computational Approaches

“…[28,29] SAMs have been intensively investigated because of their potential applications in nanotechnology. [30][31][32] We can expect that p-NBA will be able to form such monolayers at a high concentration.…”