monitoring of therapeutic treatments. Many sensing concepts have been proposed and tested in recent years to achieve this challenging mix. For these purposes, hybrid interfaces, where a self-assembled monolayer (SAM) with the desired biofunctionality is immobilized on an inorganic substrate, represent a versatile, popular platform for effective biosensors.The impressive development regarding the fabrication of functionalized DNA strands have promoted these kinds of molecules as building blocks for the development of sensing platforms. [1][2][3] Selective and reversible hybridization between complementary strands can be exploited to detect specific biomarkers, from nucleic acid target sequences (such as miRNA, [4] ctDNA [5] or viral sequences [6] ) to proteins, by employing protein-DNA conjugates. [7][8][9][10] Clearly, the outbreak of the COVID-19 pandemic has boosted the urgent need for highly sensitive, inexpensive and rapid selective recognition of SARS-CoV-2 sequences and spurred research on DNA-based detection of viral sequences. [11,12] In this work, we analyze a DNA sensing concept that is implemented through a 3-step process (Figure 1). The process is initiated with the self-assembly of single-strand DNA (HS-pDNA, 22 bases) that binds to gold through a linker (hexanethiol, C6). According to well-defined protocols, [13] this SAM is exposed to mercaptohexanol (MCH), a thiol with the same alkyl chain length of the hexanethiol linker. It has been reported that MCH co-adsorption improves DNA film organization [14][15][16][17] and increases the efficiency of the final, hybridization step [18,19] that implements the recognition of the target sequence.Literature presents varieties of methods for the recognition of target sequences through the formation of double-strand DNA (dsDNA). Some approaches involve mass sensitive methods like Quartz Crystal Microbalance (QCM) [20][21][22][23][24] or electrochemical methods. [25][26][27][28] Optical methods have been proposed that exploit, among others, colorimetric detection, [29] Surface Plasmon Resonance phenomena (SPR) [22,23,30] or combined plasmonic photothermal effects and localized SPR. [31] SPR, in particular, has been valuably employed to study surface confined DNA hybridization on a system closely related to the one under investigation here. [32] Among optical methods, Spectroscopic Ellipsometry (SE) can be advantageously employed to track changes in film Here, a comprehensive study of a label-free detection platform for the recognition of oligonucleotide sequences based on hybridization of thioltethered DNA strands self-assembled on flat gold films is presented. The study exploits in-buffer spectroscopic ellipsometry (SE) measurements, a noninvasive method sensitive to monolayer films, supported by surface mass density change measurements (Quartz Crystal Microbalance with Dissipation, QCM-D) obtained under comparable experimental conditions. SE and QCM-D allow monitoring deposition of molecular precursors and DNA chain hybridization. Combining SE measurements wi...

Porous transition metal oxides are widely studied as biocompatible materials for the development of prosthetic implants. Resurfacing the oxide to improve the antibacterial properties of the material is still an open issue, as infections remain a major cause of implant failure. We investigated the functionalization of porous titanium oxide obtained by anodic oxidation with amino acids (Leucine) as a first step to couple antimicrobial peptides to the oxide surface. We adopted a two-step molecular deposition process as follows: self-assembly of aminophosphonates to titanium oxide followed by covalent coupling of Fmoc-Leucine to aminophosphonates. Molecular deposition was investigated step-by-step by Atomic Force Microscopy (AFM) and X-ray Photoemission Spectroscopy (XPS). Since the inherent high roughness of porous titanium hampers the analysis of molecular orientation on the surface, we resorted to parallel experiments on flat titanium oxide thin films. AFM nanoshaving experiments on aminophosphonates deposited on flat TiO2 indicate the formation of an aminophosphonate monolayer while angle-resolved XPS analysis gives evidence of the formation of an oriented monolayer exposing the amine groups. The availability of the amine groups at the outer interface of the monolayer was confirmed on both flat and porous substrates by the following successful coupling with Fmoc-Leucine, as indicated by high-resolution XPS analysis.

By using AFM as a nanografting tool, we grafted micrometer-sized DNA platforms into inert alkanethiol SAMs. Tuning the grafting conditions (surface density of grafting lines and scan rate) allowed us to tailor the molecular density of the DNA platforms. Following the nanografting process, AFM was operated in the low perturbative Quantitative Imaging (QI) mode. The analysis of QI AFM images showed the coexistence of molecular domains of different heights, and thus different densities, within the grafted areas, which were not previously reported using contact AFM imaging. Thinner domains corresponded to low-density DNA regions characterized by loosely packed, randomly oriented DNA strands, while thicker domains corresponded to regions with more densely grafted DNA. Grafting with densely spaced and slow scans increased the size of the high-density domains, resulting in an overall increase in patch height. The structure of the grafted DNA was compared to self-assembled DNA, which was assessed through nanoshaving experiments. Exposing the DNA patches to the target sequence produced an increase in the patch height, indicating that hybridization was accomplished. The relative height increase of the DNA patches upon hybridization was higher in the case of lower density patches due to hybridization leading to a larger molecular reorganization. Low density DNA patches were therefore the most suitable for targeting oligonucleotide sequences.

In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale and can provide precise visual details over a wide size range, from the molecular level up to hundreds of micrometers. The nonperturbative nature, the ability to scan in a liquid environment, and the lack of need for freezing, fixing, or staining make AFM a well-suited tool for studying fragile samples such as macromolecular crystals. Starting from the first morphological investigations revealing the surface morphology of protein crystals, this review discusses the achievements of AFM in understanding the crystal growth processes, both at the micro- and nanoscale. The capability of AFM to investigate the sample structure at the single molecular level is analyzed considering in-depth the structure of S-layers. Lastly, high-speed atomic force microscopy (HS-AFM) is discussed as the evolution to overcome the limitations of low imaging speed, allowing for the observation of molecular dynamics and weakly adsorbed, diffusing molecules. HS-AFM has provided intuitive views and directly visualized phenomena that were previously described indirectly, answering questions that were challenging to address using other characterization methods.