Development of aptamer-based low-background assays for adenosine triphosphate detection

• Main Author: Peng, Yanfen.
• Other Authors: Jackie Yi-Ru Ying
• Format: Theses and Dissertations
• Language: English
• Published: 2013
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/52476
• Institution: Nanyang Technological University

Aptamers are unique single-stranded oligonucleic acids that can recognize some biomolecules with high specificity and affinity. In this study, this unique property has been applied to develop two different aptamer-based fluorescence bioassays for the detection of adenosine triphosphate (ATP). The first assay made use of an aptamer-hairpin complex composed of an ATP-specific aptamer sequence and a signaling sequence. In the presence of ATP, the aptamer specifically binded to ATP to open the hairpin structure, subsequently allowing the signaling sequence to hybridize with substrate for fluorescence signal readout. The high fluorescence background of the substrate was minimized by a Zn2+-induced energy transfer mechanism from the fluorophore to the quencher so as to significantly improve the signal-to-noise ratio. This assay demonstrated a linear detection range of ATP from 1 M to 2 mM with a detection limit of 0.15 M. To further improve the assay sensitivity for the ATP detection, a Mung Bean Nuclease (MBN) assisted signal amplification scheme was adopted in the second assay. A sensing element was designed by hybridizing the ATP-specific aptamer with two complementary sequences, one modified with fluorescein amidite (FAM) and the other with black-hole quencher (BHQ). Upon the binding of the aptamer with ATP, the double-stranded structure was denatured, and MBN then digested the single-stranded sequences to release the bound ATP from aptamer-ATP complex for binding in the next cycle. Under optimal conditions, the assay achieved a low detection limit of 7.9 nM for ATP detection, with a wide dynamic range of 10 nM to 2 mM. The two assays provided convenient means for protein detection, with potential clinical applications.