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 [1-3]. The hybridization of complementary target sequences is monitored through in situ, non-destructive, and real-time analysis.

The multiplexing detection of different oligonucleotide sequences is of great interest for differential diagnosis. To this end, we exploit AFM in a nanolithography mode to obtain micrometric platforms of thiolated DNA. Grafting is performed by removing previously chemisorbed inert alkanethiol SAMs and replacing them with short thiolated DNA molecules. Changing grafting parameters, DNA patches with different molecular densities were obtained. The analysis of images acquired in low-perturbative quantitative imaging (QI) mode highlighted the coexistence of molecular domains of different heights and thus different densities, which were not formerly observed using contact AFM imaging. By exposing the DNA platforms to target DNA (down to the nM level), all patches increased in height, indicating a successful hybridization. Comparing the height of the patches before and after hybridization showed a higher relative height increase in the less dense patches, indicating them as most suitable for targeting oligonucleotide sequences [4]. This method allows the grafting of different thiolated DNA strands onto the same substrate. Different sequences, characterized by 10 mismatches, were employed. Upon exposing the platform to different targets, a selective hybridization of specific probe DNA patches was observed, demonstrating efficient multiplexing targeting.

[1] G. Pinto, et al., 15 (2019) Soft Matter,11, 2463-2468.

[2] G. Pinto, et al., 13 (2020) Materials, 13,2888.

[3] G. Pinto, et al., 9 (2022) Adv. Mater. Interfaces, 9, 19, 2200364.

[4] S.M.C. Rotondi et al., 23 (2023) Biosensors, 9, 4557.