Detection of viral particles by using probe-gated silica nanoparticles

Early diagnosis of viral infections at point-of-care location is considered as a critical tool in the infection control. Reverse transcription-polymerase chain reaction (RT-PCR) has been a commonly employed detection methodology for virus detection, which is utmost importance for human health. But RT-PCR is a long process that has many steps. Large-scale monitoring projects are also hard because they need complicated equipment and people with a lot of experience. Therefore, the aim of this thesis study is to develop a rapid detection method working directly with samples taken from the nose and throat of SARS-CoV-2 patients. The novel method developed in this thesis based on fluorescein releasing from mesoporous MCM-41 type of silica nanoparticles (MSNPs) during hybridization between a conjugated complementary single strand oligonucleotide and SARS-CoV-2 RNA samples. For this purpose, firstly, mesoporous MSNPs were characterized by TEM to determine microstructure, BET to determine pore size and surface area and DLS to detect particle size. Then, MSNPs were loaded by fluorescein and capped by specific gene sequences probes immobilized on the surface of the nanoparticles. Three target (NSP12, NSP9 and Egene) regions selected from SARS-CoV-2 genome and tested with synthetic oligonucleotides. The test prototype was optimized in different pH and temperature conditions. Last, the human swap samples were used for verification. As a result, NSP12 gene-based detection of the SARS-CoV-2 was used to garner the best detection yield compared to NSP9 gene and E gene oligonucleotide MSNPs conjugates. The optimum target detection time was determined as 15 minutes. The basic pH damaged the structure of probe-gated MSNPs. The limit of detection with experiments using patient samples was 1.4, Relative Fluorescence Units with 84% accuracy. As a conclusion, in this thesis, a generic method based on nucleic acid-gated silica nanoparticles to detect SARS-CoV-2 in 15 minutes directly from patient swap samples was developed. This nanoparticle-based method could be improved for SARS-CoV-2 variants or any kind of different virus genome, including RNA viruses as it is faster, promising for sensitive detection, easy to operate, and less expensive than current methods.