Nanoscale Spatial Distribution of Thiolated DNA on Model Nucleic Acid Sensor Surfaces

Abstract

The nanoscale arrangement of the DNA probe molecules on sensor surfaces has a profound impact on molecular recognition and signaling reactions on DNA biosensors and microarrays. Using electrochemical atomic force microscopy, we have directly determined the nanoscale spatial distribution of thiolated DNA that are attached to gold via different methods. We discovered significant heterogeneity in the probe density and limited stability for DNA monolayers prepared by the backfilling method, that is, first exposing the surface to thiolated DNA then "backfilling" with a passivating alkanethiol. On the other hand, the monolayers prepared by "inserting" thiolated DNA into a preformed alkanethiol monolayer lead to a more uniformly distributed layer of DNA. With high-resolution images of single DNA molecules on the surface, we have introduced spatial statistics to characterize the nanoscale arrangement of DNA probes. The randomness of the spatial distribution has been characterized. By determining the local densities surrounding individual molecules, we observed subpopulations of probes with dramatically different levels of "probe crowding". We anticipate that the novel application of spatial statistics to DNA monolayers can enable a framework to understand heterogeneity in probe spatial distributions, interprobe interactions, and ultimately probe activity on sensor surfaces.

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.…”

“…Murphy et al [20] and Josephs and Ye [27] showed that this procedure produces very inhomogeneous surfaces, whereas with the use of the reversed procedure (diluent immobilized first and backfilling with DNA, procedure P3) DNA is adsorbed in the defect sites of the initial SAM of MCH, which provides a less heterogeneous but more diluted mixed SAM. In this work, we tested a third procedure, in which DNA and MCB were co-adsorbed together in a single step (procedure P1).…”

“…The presence of persistent hotspots despite the diluent treatment might be precisely related to the interactions between the DNA strands forming large-size aggregates. Josephs and Ye [27] actually suggested that the diluent may promote the lateral diffusion of individual DNA strands towards the formation of energetically favorable clusters. Moreover, these DNA clusters involve a higher number of interactions between the DNA strands and the electrode surface, and this makes them very difficult to remove both for thermodynamic and kinetic reasons.…”

A novel label-free and reusable electrochemical cytosensor for highly sensitive detection and specific collection of CTCs

“…Immunofluorescence staining procedures mainly refer to the documented protocol with minor modifications (Josephs et al, 2013;Ko et al, 2005;Schmidt et al, 1980;Wan et al, 2014). As follows: The cells were fixed with 4% paraformaldehyde for 30 min at room temperature and washed with ice pre-cold PBS, permeabilized with 0.5% Triton X-100 in PBS for 20 min (room temperature), blocked with 0.5% BSA/PBS for 1 h and then incubated with fluorescent antibody (1:10) overnight in the dark at 4°C.…”

“…As shown in Scheme 1, in the cytosensor, we inserted the capture probe in the 6-mercapto-1-hexanol (MCH) interspaces after MCH immobilized for the capture probe standing and more uniformly distributing onto the gold electrode surface (Josephs et al, 2013). The strategy could contribute to hybridize with aptamer and capture more CTCs, and then improved the sensitivity.…”