Development of highly sensitive detection systems for the diagnosis of diseases using aptamers and DNA probes

Abstract

Medical diagnosis is very important and essential to maintaining a healthy life. In vitro diagnostics have been focused on by scientists and researchers of late because it has a number of merits over other diagnostic methods. In particular, nucleic acids-based diagnostic methods are powerful and promising techniques. In Chapter 1, various types of nucleic acid-based in vitro diagnostics are introduced. These methods can be categorized into three groups according to their analytical approaches. Among these methods, aptamer-based diagnostic methods are covered in greater detail because aptamers are promising materials for diverse areas, not just as alternatives to antibodies but the core components of medical and analytical equipment. Finding a highly sensitive diagnostic technique for malaria challenged scientists in the last century. In Chapter 2, versatile single-strand DNA aptamers for Plasmodium lactate dehydrogenase (pLDH), a biomarker for malaria, are identified via the systematic evolution of ligands by exponential enrichment (SELEX). The pLDH aptamers selectively bound to the target proteins with high sensitivity (Kd = 16.8–49.6 nM). The selected aptamers were characterized using an electrophoretic mobility shift assay, a quartz crystal microbalance, a fluorescence assay, and circular dichroism spectroscopy. A simple aptasensor was also designed using electrochemical impedance spectroscopy, and Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) were selectively detected with a detection limit of 1 pM. Furthermore, the pLDH aptasensor clearly distinguished between malaria-positive blood samples of two major species (Plasmodium vivax and Plasmodium falciparum) and a negative control, indicating that it may be a useful tool for the diagnosis, monitoring, and surveillance of malaria. In Chapter 3, a simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers PvLDH and PfLDH was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with detection limits of 1.25 and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum and malaria-positive blood samples. In Chapter 4, DNA probes designed for engrailed-2 (EN2), a biomarker for prostate cancer, using homeodomain-specific binding site recognition are presented. The interaction between the DNA probes and the EN2 protein was confirmed by electrophoresis mobility shift assay and surface plasmon resonance. The affinities of the DNA probes to the target protein were determined using a fluorescence assay involving FAM-modified DNA probes and magnetic beads, and the dissociation constants were within the 61.03–98.84 nM range. A simple quantitative assay was performed to investigate the feasibility of the DNA probes. An ultrasensitive electrochemical biosensor was also developed via the electrodeposition of gold nanoparticles. The DNA probes for the EN2 protein were optimized to ensure high sensitivity and selectivity. The EN2 protein was quantitatively detected using the electrochemical biosensor, and the calculated detection limit was 5.62 fM. Finally, the specificity and applicability of the biosensor were verified using several proteins and an artificial urine medium. The impedance signals increased in the cases of EN2 and mixture containing all proteins.