Optical Nanopore Translocation Biosensor for UltraSensitive Single-Molecule Quantification of Nucleic Acids

Nanopore biosensors have become an attractive class of biosensors in recent years, especially because of the promise they hold in the single-molecule detection and quantification space. Various formats of nanopore sensors in literature have demonstrated significant potential to detect and quantify clinically relevant disease biomarkers such as proteins and nucleic acids. However, there are still some identified challenges such as lack of analyte-specific ionic current signals for electrical nanopore assays in complex biological samples, or elongated response time due to mass transport limitations. In this work, nanopore translocation and optical signal strategies have been adopted to overcome some of these identified challenges. Analyte-specific signal was ensured by deploying a functional dimeric fluorescent nanoparticle biorecognition concept, where the target nucleic acid (miRNA) via hybridization, bridges two complementary halves of DNA probes grafted on two different colours of nanoparticles. This way, each dimer nanoparticle translocation is counted as a molecule of target miRNA. This single molecule counting (via digital image analysis) makes the assay truly quantitative. Locked nucleic acid (LNA) nucleotide units were introduced between DNA probe nucleotides to stabilize the resulting DNA:RNA duplex structure, which was otherwise unstable due to low melting temperature resulting from the split-probe design. Electrical potential difference is employed to drive nanoparticles through a high-density nanopore array, enabling simultaneous monitoring of thousands of translocation events, and significantly reducing the analysis time. The developed assay was then used to detect and quantify miRNA-155 in both physiological buffer (PBS) and complex biological (cell culture) samples, reaching low femtomolar detection limit. By combining a third colour of fluorescent nanoparticles, a second miRNA target (miRNA-21) was simultaneously detected and quantified in both buffer and complex biological samples, demonstrating the multiplex detection capabilities of the system. The applicability of this assay in answering clinically relevant questions was also demonstrated by monitoring the changes in miRNA-155 levels after drug treatment of spheroids from MCF7 human breast cancer cell lines, with very promising results. In conclusion, this project has demonstrated the great potential that lies in optical nanopore sensors, being able to reach very low femtomolar detection limits with amplification-free detection and quantification of very dilute nucleic acid analytes, even in complex liquid biopsies.