DNA 압타머를 이용한 고효율 진단 플랫폼 개발 및 응용

Abstract

For several decades, a precise diagnosis has been widely studied in the world for the development of rapid, cost-effective and personalized treatment to severe diseases. To build up such a disease detection method, the biomarkers arose as novel criteria of diagnosis. The term “biomarker” refers to an indicator of the biological process that can be measured in the body to predict the incidence of disease. However, in many cases, biomarkers are often present at very low concentrations in various other proteins, resulting it more difficult to identify them. Therefore, highly efficient detection technologies are necessary to make an accurate examination for biomarkers in the early stage of the disease. Currently, clinical diagnosis with biomarkers is conducted by traditional methods such as antibody-based enzyme-linked immunosorbent assay (ELISA). However, antibodies have shortcomings to utilize such as long discovery times, huge size, poor stability, etc. Aptamers are widely used as the biomolecular probe to remedy the antibody’s shortcomings.

Aptamers are oligo-nucleic acids or peptides molecules, which usually have strong binding affinity to the specific biomarker. Each aptamer has particular 3-D structures originated from its own sequence, enabling the specific binding affinity to target biomarker over other proteins. Aptamers can be productively used to detect thousands of proteins simultaneously in platforms for multiplex discovery, where antibodies usually have problems with cross-reactivity. Through various modifications, large range of additional chemical moieties can be added at any desired position in the oligonucleotide sequences, therefore the functional groups brought the features of proteins, small molecule drugs, and antibodies together into aptamers, providing the aptamers as a most versatile reagent in medical applications. In the next chapters, newly developed diagnostic platforms using DNA aptamers are fully introduced to prove that diagnostic applications of DNA aptamers are effective.

In chapter 2, a highly sensitive detection platform for immune biomarker was developed. The rod-shaped gold electrodes with circular gold surface were used. The aptasensor specifically detected membrane-bound IL-5RA on human eosinophils according to the number of the eosinophils. We further examined the usability of soluble IL-5RA (sIL-5RA) in the serum as a novel biomarker for the diagnosis of eosinophil-related diseases. When eosinophilic inflammation occurs, the concentration of sIL-5RA in the blood is highly elevated. To diagnose eosinophilic inflammation, the aptasensor was calibrated in phosphate-buffered saline (PBS) to detect sIL-5RA. The detection limit of the aptasensor in PBS was 1.69 pg/mL, which is lower than that of commercially available ELISA kits. The dynamic range of the aptasensor was 10–10,000 pg/mL, with high specificity over other serum proteins and interleukin family members. The aptasensor was capable of distinguishing between patients with eosinophilic asthma (EA) and normal controls (NCs) based on serum sIL-5RA levels. Consequently, the fabricated aptasensor will contribute to the development of novel diagnostic systems for IL-5RA-related diseases.

In chapter 3, an ultra-sensitive detection system for cardiac biomarker was developed. In this study, a plasmonic enzyme-linked aptamer assay (ELAA) was developed to enable the quantification of cTnI with a wide detection range. This detection system involved cTnI specific aptamers that were identified in a previous study and signal amplification was performed using the plasmonic effect of gold nanoparticles (AuNPs), which depends on the size and shape of the particles. Based on simple UV-Vis spectroscopy, the designed plasmonic ELAA showed ultra-sensitive performance in the analysis of cTnI with a wide detection range 1 fM–1 nM (detection limit: 0.52 fM), which has not been demonstrated previously.