Design of nanomaterials for oligonucleotide detection

Abstract

Nanoparticles are considered a powerful tool for novel applications ranging from imaging and sensing to therapy. In this thesis, gold nanoparticles functionalised with DNA strands were used for the detection of target oligonucleotides in live cells while upconversion nanoparticles functionalised with DNA strands were used for the detection of target oligonucleotides in a tube. Gold nanoparticles functionalised with synthetic DNA oligonucleotides were used to identify and sort skeletal stem cells upon specific mRNA detection. Human bone marrow stromal cells include a population of skeletal stem cells, with the capacity to differentiate along the osteogenic, adipogenic, and chondrogenic lineages. According to current techniques, the isolation and enrichment of skeletal stem cells from human tissues face challenges when there is no specific skeletal stem cell marker. DNA functionalised AuNPs were able to detect skeletal stem cells based on endocellular mRNA expression and to rapidly sort these cells from human bone marrow. This is a significant approach for tissue engineering as skeletal stem cells can be harnessed to help bone regeneration. The enhancement of the endosomal escape of DNA functionalised gold nanoparticles via the incorporation of a cell-penetrating peptide has been studied in order to maximize the concentration of gold nanoparticles that can detect mRNA and therefore reduce their total amount when incubating with cells. Two different approaches for the functionalisation of gold nanoparticles’ surfaces with peptides are presented. In the first case, gold nanoparticles were functionalised with thiol terminated DNA strands followed by functionalisation with cysteine terminated cell-penetrating peptides. In the second instance, the peptide was conjugated on the 5’ end of the DNA strand. Then, the gold nanoparticles were modified with these conjugate sequences. All the above probes exhibited similar stability towards degradation by endocellular enzymes and similar specificity towards the detection of specific mRNA targets. Finally, the fabrication of an oligonucleotide sensor was developed based on the emissive optical properties of oligonucleotide-coated lanthanide-doped upconversion nanoparticles and the quenching ability of two-dimensional materials; MoS₂, WS₂ and graphene oxide. In the first system, it was investigated the development of an upconversion nanoparticles sensor that detected polyA sequences and used the twodimensional materials, MoS₂ and WS₂, as quenchers. In the second system, the fabrication of a DNA sensor for SARS-CoV-2 oligonucleotide detection was demonstrated while in this case, graphene oxide was the quencher. In both cases, monodisperse upconversion nanoparticles were functionalised with single-stranded DNA. In the presence of a complementary oligonucleotide target and the formation of double-stranded DNA, the upconversion nanoparticles could not interact with two-dimensional materials, thus retaining their fluorescence properties. The high sensitivity and specificity of this sensor to detect specific target oligonucleotides were also monitored.

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Identifiers

ORCID for Konstantina Alexaki: orcid.org/0000-0002-8702-2696

ORCID for Antonios Kanaras: orcid.org/0000-0002-9847-6706

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