Controlling conformational and mechanical properties of single-stranded DNA (ssDNA) nanobrushes is crucial for the design of new, miniaturized DNA-based functional biosensors. In particular, counterions diffusion and binding affinity to DNA impact on ssDNAs curvature and flexibility and modify their binding properties. In order to highlight the role of cation electrostatic screening and molecular crowding on the conformational stability of DNA brushes, we propose here to use atomic force microscopy (AFM) and AFM-based lithography to create ssDNA assemblies of variable density and to analyze their collective response to changes of ionic strength. We confined ssDNA brushes with controlled surface densities within a biorepellent self-assembled monolayer. We then monitored the topographic brush height variation upon changing salt type (NaCl, KCl, CaCl 2 , and MgCl 2 ) and concentration inside the liquid cell. We show that the measured height is related to scaling law of salt concentration, in agreement with the theory of polyelectrolyte brush. We find the same scaling exponent α = −1/6 for the different density regimes exploited. Using this scaling model to fit our experimental data, we quantified structural parameters such as the average internucleotide distance (d) for ssDNA brushes of different and estimated surface density (σ), featuring a strong dependence of d on different salts species. ■ INTRODUCTIONIn the past decade single-stranded DNA (ssDNA) monolayers (or ssDNA brushes) immobilized on solid supports have attracted vast attention due to their interesting physical and chemical properties. Such systems have found applications in biomolecular detection as DNA-based microarray 1−4 and protein biosensors, 5,6 which nowadays are used for identification of genetic diseases 7−9 and for the characterization of gene expression profiles.The assembly of ssDNA brushes on a solid support is commonly assumed to be aided by some structural water and cation species, which shield the backbones of adjacent, vertically oriented, DNA molecules creating intermolecular channels with subnanometer diameter. Understanding the properties of ssDNA brushes and their response to different factors (e.g., counterion species, pH, humidity, pressure, and temperature) is therefore crucial for optimization of DNAbased biosensors. 10−12 In particular, counterions play an important role in stabilizing the electrostatic charge of the DNA backbone and impact on its curvature and flexibility. 13,14 Counterion size, valence, and concentration affect the efficiency of DNA hybridization 15,16 and have a strong effect on DNA−protein interactions in the context of replication and transcription. 17 While many studies based on different techniques (e.g., optical tweezers, 18−20 atomic force microscopy (AFM), 21,22 and a combination of small-angle X-ray scattering (SAXS) with single molecule Forster resonance energy transfer (FRET) 23 ) have discussed the effect of counterions on DNA molecules both in bulk and at the single-molecule level, still lit...

Single-stranded DNA (ssDNA) brushes, in which ssDNA oligomers are tethered to surfaces in dense monolayers, are being investigated for potential biosensing applications.

High-density monolayers (HDMs) of single-strand (ss) DNA are important nanoscale platforms for the fabrication of sensors and for mechanistic studies of enzymes on surfaces. Such systems can be used, for example, to monitor gene expression, and for the construction of more complex nanodevices via selective hybridization with the complementary oligos dissolved in solution. In this framework, controlling HDM hybridization is essential to control the final properties. Different studies demonstrate that at the typical density of ≈10(13) molecules per cm(2) no more than ≈30-40% of the HDM ssDNA is successfully hybridized. Until now, however, the origin of the HDM hybridization limit has remained unclear. In this work, molecular dynamics (MD) simulations of HDM systems with variable hybridization reveal that, independently of other experimental parameters, the effective hybridization for a HDM of this density is intrinsically limited by molecular and electrostatic crowding. A detailed structural analysis of the HDM model shows good agreement with our atomic force microscopy (AFM) experiments, and provides further insight into the steric hindrance behaviour and time-resolved surface topography of these nanostructured systems. The explicit relationship proposed between structural crowding and limited HDM hybridization offers a rationale to control the final properties of HDM-based nanodevices.