การสังเคราะห์พีเอ็นเอแอเรย์บนกระดาษกรองดัดแปรพื้นผิว เพื่อใช้ตรวจสอบลำดับเบสของสารพันธุกรรม

• Main Authors: เกรียงศักดิ์ ฝ้ายเครือ, กชกร ศุภบวรสถิตย์
• 其他作者: ธีรยุทธ วิไลวัลย์
• 格式: Senior Project
• 語言: Thai
• 出版: จุฬาลงกรณ์มหาวิทยาลัย 2014
• 在線閱讀: https://digiverse.chula.ac.th/Info/item/dc:9808
• 機構: Chulalongkorn University

Cellulose-based filter paper is an attractive material for immobilization of biomolecular probes for sensing applications due to its general availability, biocompatibility, and possibilities for a wide range of surface functionalizations. In this work, pyrrolidinyl peptide nucleic acid with a rigid D-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA, will be simply referred to as PNA) is immobilized on to a cellulose surface to be used as a probe for DNA sequence determination. First, a dialdehyde-modified cellulose (DAC) was prepared by partial oxidation of cellulose paper with sodium periodate in the presence of lithium chloride. The presence of the aldehyde group was revealed by a color test with 2,4-dinitrophenylhydrazine. The PNA probe was next immobilized by reductive alkylation in the presence of sodium cyanoborohydride. By employing a fluorescein-labeled acpcPNA probe, successful immobilization of the PNA was demonstrated. Next, the ability of the immobilized PNA probe to capture the DNA target from the sample was confirmed using unlabeled PNA probe and fluorescein-labeled DNA. As low as 0.35 pmol of fluorescein-labeled DNA could be detected under UV light, suggesting the potential of this aldehyde-based immobilization strategy in fabrication of PNA arrays. Furthermore, an enzyme-based colorimetric detection was also developed in order to improve the sensitivity and avoid the expensive fluorescence labeling of the DNA target. Upon hybridization between the immobilized PNA probe and biotinylated DNA target followed by treatment with streptavidin-horseradish peroxidase conjugate, a colorimetric reaction was achieved by treatment with o-phenylenediamine/H2O2 which allowed visual detection of the yellow product formed. The cellulose-based DNA sensor with immobilized PNA probe and enzyme-based colorimetric detection is sensitive enough to detect sub-picomole amounts of synthetic DNA targets with sufficient specificity to discriminate between complementary and single base mismatch DNA targets.