Here, a comprehensive study of a label-free detection platform for the recognition of oligonucleotide sequences based on hybridization of thiol-tethered 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 with QCM-D data paves the way for quantification of the assay through the possible calibration of SE data. Optical measurements also demonstrate the selectivity and recovery properties of the sensing platform. Broadband SE measurements are interpreted by means of an effective optical model. The model, complemented by information on film thickness (scanning probe nanolithography), and surface composition (monochromatic X-ray Photoemission Spectroscopy, XPS), enables a clear spectral identification of UV DNA resonances and the formation of the thiolate interface with gold. Spectroscopic validation of the hybridization is complemented by employing labeled target strands. The influence of hybridization on UV resonances and optical thickness of the DNA film is discussed in the light of hypochromism, through comparison with QCM-D data.

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.

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.

We employ Spectroscopic Ellipsometry (SE) in the Kretschmann—Raether (KR) configuration (KRSE) to characterize a DNA‐functionalized platform designed for detecting specific viral oligonucleotide sequences. KRSE merges SE with Surface Plasmon Resonance, improving sensitivity to subtle interfacial phenomena. The sensor consists of a self‐assembled monolayer of thiolated DNA probes mixed with mercaptohexanol (MCH) spacers to promote upright probe orientation. The system is evaluated for its ability to selectively recognize the RdRp‐Helicase sequence of SARS‐CoV‐2, effectively discriminating it from the still circulating SARS‐CoV HKU strain. KRSE spectra exhibit pronounced redshifts (δλ) of the resonance as a function of target concentration. The formation of the probe layer and, in particular, the deposition of MCH, induce a deeper minimum in Ψ vs λ data at resonance. No further variation of the value of Ψ at resonance is observed in sensing/regeneration/sensing cycles. Optical modelling indicates that δλ redshifts are determined by increases in optical thickness of the adsorbed layer, while the Ψ changes at resonance derive from the formation of a sub‐nanometric Au‐thiol interfacial layer. This interface is modelled using a Bruggeman effective medium approximation, following previous standard SE experiments. Analysis of δλ shifts allows to derive a calibration curve with a nanomolar detection limit, and enables precise assessment of sensor specificity, outperforming previous conventional SE measurements on the same system.

Coupling spectroscopic ellipsometry (SE), quartz crystal microbalance with dissipation (QCM-D), X-ray photoemission spectroscopy (XPS), and atomic force microscopy (AFM), we developed a multi-technique approach to characterize the surface immobilization of probe DNA strands, as a tool for the design of a DNA-based biosensor for the detection of disease-related oligonucleotide strands