Bio-organic/inorganic interfaces for biosensor design

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), first reported at the end of 2019, triggered extensive research into developing functional biosensing platforms for the early diagnosis of viral infections. In February 2020, the World Health Organization (WHO) declared SARS-CoV-2 a pandemic, as its global impact far exceeded that of previous coronavirus outbreaks, such as SARS-CoV and MERS-CoV. During the pandemic, the primary diagnostic methods for SARS-CoV-2 were Reverse-Transcription Polymerase Chain Reaction (RT-PCR), which amplifies and detects viral RNA, and antigen tests, which identify viral proteins in nasal or throat swabs. However, these methods had notable limitations: RT-PCR required long processing times, was costly, and had limited accessibility, while antigen tests were generally less sensitive. Consequently, the need for fast, reliable, and sensitive diagnostic techniques became increasingly urgent. In this context, bio-sensing interfaces, a class of biosensors that leverage high binding specificity, emerged as promising tools for detecting nucleic acids, enzymes, and antibodies. These sensors are based on the fabrication of hybrid interfaces, where sensing molecules are immobilized onto a surface. A widely adopted approach involves self-assembly, a spontaneous molecular process that leads to the formation of ordered structures known as Self-Assembled Monolayers (SAMs). Among the molecules used for SAM formation, alkanethiols are particularly well-suited for gold substrates due to their strong thiol (-SH) affinity for gold surfaces. By functionalizing alkanethiols with specific chemical groups, biomolecules such as DNA, proteins, or antibodies can be anchored to the surface without denaturation, making SAMs highly suitable for biosensing applications. For viral RNA detection, a single-stranded DNA (ssDNA) probe complementary to the target sequence can be used. In the case of SARS-CoV-2, the RNA-dependent RNA-polymerase (RdRp) Helicase sequence remained highly conserved across all viral mutations, making it an excellent candidate for detection. Considering these aspects, this thesis focuses on the fabrication and characterization of SAMs composed of thiolated single-stranded DNA (probe DNA, pDNA) and their hybridization with the complementary target DNA (tDNA), specifically the RdRp/Helicase sequence of SARS-CoV-2. Given the complexity of this system, a multi-technique approach was employed for characterization. Atomic Force Microscopy (AFM): AFM was employed as a nanolithography tool. Nanoshaving experiments provided insights into SAM thickness under different conditions and for various DNA sequences. Additionally, AFM nanografting was used to pattern the sensing surface with distinct probe DNAs, enabling the parallel detection of multiple sequences. Quartz Crystal Microbalance with Dissipation (QCM-D): This technique was used to monitor molecular deposition and evaluate molecular surface coverage. Spectroscopic Ellipsometry (SE): This method has been implemented in both external reflection (standard SE) and total internal reflection configuration (TIRE). Ellipsometry data provided insights into the optical properties of the DNA SAMs, including the UV absorption of immobilized DNA. As discussed in the thesis, given the ultrathin nature of DNA SAMs, information on film thickness retrieved from AFM allowed for the accurate interpretation of ellipsometry data. The analysis of both standard SE and TIRE data provided information of interface effects related to the strong coupling between thiolated molecules and gold. This multi-technique approach enabled the study of key biosensing properties, including sensitivity, selectivity, and reusability, essential for the development of an effective and reliable biosensing platform.