Advanced Technologies in Rapid and Multiplex Detection of Nucleic acid

Nucleic acids are key macromolecules of living organisms transferring genetic inheritance from one generation to the next. From how a living individual is created to how it interacts with external factors, all and all, can be found in the nucleic acid sequences inside every single cell of every organism. Therefore, the analysis of nucleic acids sequences is a critical capability for cancer and pathogen diagnoses, genotyping, and disease monitoring. To date, numerous methods have been used to detect both characterised and uncharacterised mutations and sequence variations. However, the detection of low amounts of mutant genes in the presence of high levels of wildtype sequences is still a challenge, and existing technologies has room for improvement. The classical approaches for nucleic acid detection and analyses mainly include DNA sequencing and the polymerase chain reaction (PCR). Although these methods with high analytical performance and reliability have facilitated the interrogation of the nucleic acids, some key obstacles such as the need of labelling, high costs for routine clinical use, slow turnaround time for giving results, the complexity of operation, and the inability to dually detect the genetic mutation in one step have limited their applications. To avoid drawbacks of the traditional approaches, a number of chip-based methods leverage electrochemical readouts or microfluidics to identify nucleic acids. However, there is still an unmet need for a less complex, rapid, low-cost, sensitive and accurate method to enable nucleic acid analysis even in resource-poor settings. The overall objective of this PhD thesis is to develop simple, inexpensive and accurate platforms for nucleic acid evaluation. To achieve the aforementioned goal, the first attempt was to develop a lab-on-a-chip platform for cancer diagnosis by detection of circulating tumor nucleic acids (ctNAs) in plasma samples of cancer patients. ctNAs are fragmented DNA released from cancerous cells and tumours into the bloodstream of patients with cancer. Tumour-specific (epi-)genetic alterations in ctNAs are assumed to reflect tumour burden and could be of high value for cancer diagnosis, prognosis, and management. In the first part of the thesis, I developed a new electrochemical assay for the detection of FGFR2:FAM76A fusion gene in ctNAs extracted from ovarian cancer patients. The assay was based on the high electrocatalytic activity of a new class of superparamagnetic graphene-loaded iron oxide nanoparticles. Electrochemical detection demonstrated a limit of detection (LOD) as low as 1.0 fM, high specificity and excellent reproducibility. In the second part of the thesis, I designed and developed a real-time and quantitative PCR system for microbial source tracking (MST) in water samples. MST is a DNA-based technology that enables water-quality managers to identify sources of faecal pollution in environmental waters. Most of the MST methodologies typically require specialized and costly equipment, elaborated and time-consuming operations as well as trained personnel. Here, a simple, low-cost, and sensitive platform was implemented on a microfluidic array chip. The array was successfully used for the real-time PCR-based multiplex detection of three human-associated MST markers (H8, Gen bac III, UidA). The PCR mixture was loaded into an array of channels in a single step utilising capillary filling without the need for liquid handling instruments. The array was then integrated with our custom-made thermal cycling and optical detection system. By employing the fabricated platform, the LOD of 71.8 DNA copies/μL was achieved for Gen bac III sequence. In summary, we introduced a sensitive, simple and economical real-time and quantitative PCR system for MST in water samples. In a further study, I investigated how a nucleic acid amplification setup can be miniaturised. To reach this goal, I utilised liquid marbles as an ideal biochemical microreactors for targeted amplification of the NAs. Liquid marbles are formed by encapsulating microscale volume of liquid with a thin layer of hydrophobic particles. Miniaturization of the nucleic acids (NAs) amplification process inside a liquid droplet provides several advantages upon routine methods, such as reducing reagents consumption and contamination possibility, easy handling of liquids, eliminating the usage of disposable plastic consumables for carrying out biochemical reactions. However, one of the major concerns in liquid marble applications is the high rate of evaporation through the porous walls during the thermal cycling step. To eliminate the evaporation, I used core-shell beads synthesized from a composite liquid marble as a NAs amplification micro reactor comprising two non-miscible liquid droplets forming a spherical shape and a coating of hydrophobic powder. The shell liquid was then polymerised into a solid after exposure to blue light, converting the liquid marble into a core-shell bead. Fabricated core-shell beads were extended to explore their potential as a versatile bioreactor for phylogrouping of the E.coli strains. In general, this platform provided easy manipulation and storage of sample, elimination of the evaporation, and sample protection from possible external contamination. Moreover, this simple and effective method presented a sensitive and inexpensive way to track NAs. In conclusion, this research endeavour presents a step forward towards the adaption of the selected group of tools and technologies, for the development of assays that can be applied as powerful alternatives to conventional tools used in molecular diagnostic. These technologies have the potential to revolutionise the NAs-based diagnostic approaches, by providing sensitive, rapid, accurate, and inexpensive platforms for point of care devices and in-field tests.