Development of nano-engineered nucleic acid testing methods for rapid pathogen detection and disease diagnosis

Abstract

For diagnosing infections, a simple and facile assay which can be directly visualized with minimal technical requirements is in great need. Nucleic acid analytes is one of the most widely used for identification of molecular targets as biomarker in biomedical research. A rapid and simple detection method for nucleic acid anlaytes allows rapid diagnosis of disease and subsequent treatment. Nucleic acid amplification techniques are used for molecular biology research and medical diagnostics to specifically detect and analyze small amounts of genetic targets. Especially, the improved detectable signal can be achieved through integrated with nanotechnology. This paper is organized as follows. In Chapter 2, we introduce a fluorescent nanosensor utilizing carbon dot nanoparticles that form visible precipitates in the presence of target DNA. Carbon dot nanoparticles were fabricated by microwave pyrolysis of polyethylenimine, which emitted strong photoluminescence and could form precipitates when added with target DNA oligonucleotides. The precipitates could be easily visualized by UV illumination, and data could be acquired as images using a smart phone which were analyzed for quantification. This carbon dot-based assay allowed fluorescent sensing of target oligonucleotides with various sizes, and visualization even with minimal amount of DNA (~100 pmol). Finally, the assay could be applied as a nanosensor platform for detecting bacterial DNA for the antibiotic resistance gene KPC-2 from Klebsiella pneumoniae. This method provides a simple technique for detecting molecular targets, showing wide applicability for diagnostics on the bedside or point-of-care testing. In Chapter 3, we propose a simple paper-based fluorescent sensor for the detection of SARS-CoV-2 using isothermal amplification method, RCA. The fluorescent particles of solution on the filter paper are radially transferred to generate the fluorescent ring patterns by capillary phenomenon. During the RCA amplification process, a micron-sized ssDNA was produced in which a certain sequence is repeated, and this DNA was labeled through hybridization with probe-dye, which has the complementary sequence of RCA products, allowing to be concentrated fluorescent signal in the central droplets on the filter paper. The target can be detected simply by fluorescent ring pattern formed under a UV lamp after the droplet is completely spread within a few minutes. Furthermore, the assay is possible to detect the multiple targets by designed the different padlock templates and probe-dyes with various emission wavelengths. The assay can be a useful and easily applicable diagnostic tool for point-of-care detection of various infectious diseases such as COVID-19, bacterial infections. We expect that the diagnostic platform, which is simple, easy-to-use compared to the existing technologies, developed in this study is possible to advance for self-diagnosis in the future through low-cost analysis equipment, allowing to change the place of diagnosis from hospital to home.