Study on the development of the two periostin-specific ssDNA aptamers and the fabrication of the fluorescence nanosensor for rapid, simple, and sensitive detection of periostin

Abstract

As the diagnosis and treatment of various diseases become possible through advances in medical technology, the quality of human life is improving. In addition, as healthcare systems capable of monitoring individual health and disease states have been developed, rapid diagnosis and treatment of diseases are becoming possible. For these reasons, the development of inexpensive, convenient, and sensitive diagnostic devices is demanded. Biomarkers are indicators that can detect changes in the body and provide in diagnosing and verifying the prognosis of diseases. In addition, they exist in small amounts in the body. Therefore, the development of highly sensitive detection platforms is necessary to detect them. Till now, many detection systems have been developed using antibodies and have high sensitivity and specificity for detecting the target molecules. However, conventional antibodies-based detection methods have several disadvantages, including strict storage and usage, complicated to operate, expensive, time-consuming, and requiring experts. Aptamers, known as chemical antibodies, have superior advantages over antibodies, such as no batch-to-batch variation, mass synthesis with low costs, easy chemical modification, and usage for various applications. Therefore, they have been utilized in various fields, including imaging, sensors, and cargo for drugs. In this study, the development of two periostin-specific ssDNA aptamers to replace antibodies and the development of the novel fluorescence nanosensor for periostin detection using fluorophores, aptamers, and metal nanoparticles are introduced. The first study (Chapter II) introduces the development of the two periostin-specific ssDNA aptamers and the verification of their simultaneous binding properties to periostin. Two ssDNA aptamers that specifically bind to periostin were developed using the systematic evolution of ligands by exponential enrichment (SELEX) method after obtaining excessive amounts of recombinant periostin protein through a bacterial expression and purification process. The selected two ssDNA aptamers exhibited good binding affinities to periostin through a magnetic bead-based fluorescence assay, and their binding affinities were further improved via the sequence-optimization process. Meanwhile, the ability of the two aptamers to simultaneously bind to periostin without interfering with each other was verified via direct and sandwich enzyme-linked oligonucleotide assay (ELONA) methods. Furthermore, the sandwich ELONA was optimized to verify whether periostin can be detected via the simultaneous binding phenomenon of the two aptamers. Indeed, this assay system detected periostin in buffer and spiked human serum conditions, achieving detection limits of 0.32 nM and 5.3 nM within 3 hours, respectively. In addition, the aptasensor showed excellent specificity by selectively detecting only periostin among other blood proteins under buffer conditions. Therefore, this study provides the possibility of developing aptamer-based periostin detection methods that can detect periostin cost-effectively, rapidly, and sensitively using aptamers, a new branch of molecular probes that can replace antibodies. The second study (Chapter III) introduces the development of a one-shot novel fluorescence nanosensor that can rapidly, label-freely, and sensitively detect periostin using two aptamers, fluorophore, and metal nanoparticles. The nanosensor quantitatively detects periostin following two mechanisms; the increased proximity between metal nanoprobes by the aptamer-protein interaction and the regeneration of the quenched fluorescence by metal nanoparticles. When periostin is present in the solution, the two nanoparticles bind to periostin in a core-satellite form by simultaneous aptamer-periostin binding. At this time, the quenched fluorescence of the nanosensor is regenerated by the metal-enhanced fluorescence (MEF) effect of the silver nanoprobes induced by the increased proximity of the two nanoprobes. Therefore, the nanosensor can detect periostin in proportion to the regenerated fluorescence intensity. To fabricate the nanosensor, gold nanoprobes were constructed by fluorophore molecules (rhodamine-b isothiocyanate, RiTC), PL2trunc aptamer, and gold nanoparticles, and silver nanoprobes were fabricated with PL5trunc aptamers and silver nanoparticles. In addition, the feasibility of the detection performance of the nanosensor was investigated by varying the concentration of the gold and silver nanoprobes. After optimization, the established nanosensor detected periostin within 30 min and without additional signal amplification and washing processes, achieving a detection limit of 106.8 pM in buffer conditions. Moreover, the nanosensor can detect only periostin, among other blood proteins. Furthermore, the nanosensor detects periostin in spiked human serum conditions with a detection limit of 463.3 pM, indicating the clinical applicability of the nanosensor. Therefore, the proposed nanosensor can identify patients with various diseases and classify the disease severity by periostin concentrations. In addition, this platform can provide a practical method for on-site medical diagnosis because the aptamers can be easily replaced other biomarker probes. Aptamers have been widely used in various fields, such as disease diagnosis, bioimaging, and drug delivery. In addition, they have been widely applied as molecular probes to replace antibodies due to their advantages, including no batch-to-batch variation, synthesis in large quantities at low cost, easy chemical modification, and low immunogenicity. Nanoparticles also have been used in many research fields for their strengths, including large surface area-to-volume ratio, simple synthesis, easy surface modification, and fluorescence quenching and enhancement by the FRET and MEF effect as the distance with the fluorophores. Therefore, the development of biosensors and molecular probes through the fusion of aptamers and nanoparticles is expected to overcome the limitations of existing diagnostic methods and further contribute to fields requiring on-site medical diagnosis and other research fields.