Studies on Biosensor Development Using Aptamers for Diagnosis of Flavivirus Infectious Diseases

Abstract

Dengue fever (DF) and yellow fever (YF) are caused by mosquitoes carrying the flaviviruses, dengue virus and yellow fever virus. They are mainly transmitted in tropical and subtropical regions. The infectious areas continue to expand and sporadic re-emergence occurs up to date, leading to an important global health problem. Moreover, vaccine supply problems or inadequate medical controls in many epidemic countries bring the necessity of efficient diagnostic technologies. In addition, DF and YF are highly difficult to differentiate in diagnosing because they are derived from the same genus as Flavivirus with a close genetic relationship. The secondary cross-infection with each other is fatal by causing severe symptoms. Thus, new diagnostic methods are required to overcome the cross-reactivity against other flaviviral infections. This thesis introduces new ideas of disease-related biomarker NS1 detection for DF and YF and provides a great insight into the use of aptamers as future biosensors to substitute the conventional diagnostic methods. Aptamers are oligonucleotide molecules that form a unique three-dimensional structure that can specifically bind to any targets. They have useful advantages such as target-specific interactions, a wide range of target molecules, stable structure maintenance with folding properties, and low production costs. It is suggested that the aptamers become useful probes as an alternative to antibodies, their drawbacks such as long time-consuming synthesis, reduced activity after modification, and low stability with short shelf-life in molecular diagnostics. Chapter I introduces the fundamental background of this thesis. The current issues and severity of DF and YF are presented in Chapter I.1.1, and diverse existing diagnostic methods are summarized in Chapter I.1.2. In addition, the necessity of developing new techniques for diagnostics is mentioned in Chapter I.1.3. Chapter I.2 covers a basic overview for understanding the development of DNA aptamer-based biosensors, the main theme of Chapter II and III. Lastly, the rapid and simple one-shot aptasensor for DF diagnosis and the first ELONA development for YF diagnosis are introduced in Chapter II and III. A new G-quadruplex (GQ)-based fluorescent aptasensor using one-shot detection of NS1 for dengue fever diagnosis was proposed in Chapter II. To design the aptasensor, Dengue virus-derived NS1-binding aptamer (DBA; Kd = 38.53 nM) that forms a GQ structure was developed. The DBA underwent structural destruction induced by nonstructural protein 1 (NS1), then it was attempted to utilize for optical sensing. Fluorescent detection of the NS1-dependent structural change was performed by applying a fluorescent dye; 6-carboxyfluorescein (FAM) was attached to the 5' end of DBA (5' FAM-DBA). The 5' FAM-DBA could quantitatively detect the fluorescence quenching caused by guanines upon NS1 binding, which was feasible for dengue fever diagnosis. To understand the structural and fluorescent characteristics, the interaction between NS1 and DBA was analyzed. The results suggested that the central G-tracts were rearranged to adjacent toward the FAM, and that the structure of 5' FAM-DBA bound to NS1 was expected to form base-pairing. Finally, the aptasensor with 5’ FAM-DBA was established after optimization, contributing to effective and rapid detection. The aptasensor achieved low detection limits of 2.51 nM in buffer and 8.13 nM in serum. Additionally, it was verified the high specificity using other proteins including yellow fever proteins, and it has the potential to overcome the cross-reactivity with other flaviviruses. Also, the stability of the DF aptasensor was confirmed to give practical usability. The novel GQ-based fluorescent aptasensor for rapid and simple dengue fever diagnosis, which could provide the possibility for use in practical applications, was developed. Chapter III presents the first enzyme-linked oligonucleotide assay (ELONA) for YF diagnosis using developed two aptamers, YFns1-4 and YFns1-31. The aptamers were selected to specifically bind to nonstructural protein 1 (NS1), which is secreted at a high concentration after infection. The binding affinities of YFns1-4 (Kd = 113.41 nM) and YFns1-31 (Kd = 24.01 nM) to the NS1 were confirmed. The aptamers did not interfere with each other on binding to the NS1, so we applied them in sandwich ELONA. The best detection sensitivity was obtained using the combination of YFns1-31 as capture aptamer and YFns1-4 as detect aptamer. The results of the direct ELONA with each YFns1-4 and YFns1-31, yielded great absorbance intensity and a broad detectable range of NS1, could support the performance of the sandwich assay. The assay achieved a desirable low detection limit of 0.85 nM that is useful within the clinical range and colorimetric change that allows visualization with the naked eye. The sandwich assay was greatly performed in 5% human serum solution, indicating clinical applicability with excellent reproducibility and recovery. In addition, the assay was not affected by other proteins including NS1 derived from dengue virus; this result confirms the potential of ability to distinguish YF from other flaviviral infections. Here, we developed a novel sandwich ELONA based on the NS1-specific aptamers for YF diagnosis; it had outstanding analytical performance and was implemented in a short time (< 3 h), providing the possibility for practical use.