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

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.

通过电化学研究结合荧光成像表明，由单链或双链DNA自组装单分子层（SAMS）修饰的金表面的制备会产生不均匀的表面堆积密度。通过硫醇盐DNA的直接吸附（随后用巯基己醇（MCH）钝化）所制备的修饰表面导致一些区域被DNA SAM单分子层覆盖，而其他区域则被大的DNA颗粒覆盖。从这些区域测量到的荧光强度差异显著。更重要的是，观察到荧光强度随电化学电位的区域变化：大的聚集体显示出与单分子层覆盖区域明显不同的荧光强度调制。对荧光标记DNA的电化学解吸和检测提供了表面完全修饰的明确证据。这些研究对使用DNA SAMS的生物传感器/生物芯片开发具有影响。用于制备DNA SAMS的方法的一种改进产生了一种明显不同的表面，其聚集体少得多，荧光强度的电调制更显著，尽管DNA表面密度低得多（约为最大理论覆盖率的1%）。这种形成修饰表面的方法与目前公认的做法相比具有明显优势，并强调了使用像电荧光显微镜这样的原位成像工具研究传感器表面非平均性质的重要性。