Towards a DNA-based biosensor: a multi-technique study for sequence recognition

The challenge to defeat diseases starting from a rapid diagnosis has been relighted by the last pandemic due to SARS-CoV-2. The development of biosensors able to recognize molecular adsorption on surfaces plays a crucial role in this issue, allowing for example to detect the presence of disease biomarkers, like specific nucleic acid sequences or antibodies, in solution. Currently, viral RNA detection relies almost exclusively on polymerase chain reaction (PCR), requiring an expensive and complex process, or on fluorescent methods using labels that could interfere with the hybridization process. We exploited spectroscopic ellipsometry (SE) label-free experiments to detect UV-Vis molecular absorptions at the monolayer level in order to develop biosensing platforms. Spectroscopic ellipsometry is a non-perturbative and extremely sensitive method for the analysis of ultrathin molecular films. Changes in polarization of a light beam upon reflection from the sample surface are detected, providing information, both quantitative and molecular-specific, on film thickness and optical properties. In fact, SE difference spectra, obtained as the difference between the spectra acquired after the molecular deposition and the spectra acquired on the bare substrate, clearly show molecular absorption fingerprints in the UV-Vis-NIR spectral range. We focus on an optical DNA-based biosensor, where DNA strands immobilized on gold can detect specific SARS-CoV-2-related RNA sequences through nucleic acid hybridization. DNA-based biosensors are an excellent option to develop micro-nano devices which are reusable thanks to the denaturation property of nucleic acids. DNA self-assembled monolayers are studied in situ and in real time through a multi-technique approach, in order to optimize the experimental protocol and design the cheaper and more efficient biosensor. The tuning of several parameters, like ssDNA immobilization time, concentration of molecular spacer solution and solution ionic strength, allowed us to highlight the subtle interplay of these factors in the DNA self-assembly process. Upon hybridization, changes in film thickness (AFM nanolithography, SE), molecular UV-Vis absorption (SE), coverage (XPS, QCM-D) are detected. In particular, SE allows to recognize the double-helix formation in situ, in a non-destructive and extremely fast way, and to detect DNA hypochromism at the monolayer level, for the first time to our knowledge. In conclusion, an accurate optical SE model, supported by AFM thickness data, allowed us to optically characterize ssDNA and dsDNA films in the whole probed wavelength region, providing a new tool for nucleic acid sequence recognition.