What is an Aptamer?
An aptamer is a stable DNA or RNA molecule that has been selected from random pools based on it’s ability to bind with high affinity to target molecules such as small organics, proteins, and even entire cells. Unlike antibodies, aptamers are selected in vitro, are capable of higher specificity and affinity, can tolerate pH and temperatures that their protein counterparts cannot. These features allow them to be highly specific detectors and to be stored at room temperature with a much longer shelf life than antibodies.
How do fluorophores and quenchers work?
A fluorophore is a chemical group that fluoresces when exposed to a specific wavelength of light. Fluorophores can be attached to oligonucleotides to create a fluorescent transducer. Popular green, red, and blue fluorophores used in direct labeling are fluorescein, rhodamine, and amino methyl coumarin, respectively. Fluorescein is a fluorescent chemical compound that emits 521 nm green light when it is excited with red light. Dabcyl is a dark quencher, which is a compound that absorbs excitation energy and re-emits the energy as heat. Dabcyl is a weak dark quencher that absorbs green light spectra when in close proximity to their source. Thus, the pairing between fluorescein and dabcyl are fitting. There are additional solutions for quenching available. One such solution are gold nanoparticles, which are essentially very large dark quenchers. Positioning a fluorophore directly adjacent to a gold nanoparticle will cause the fluorescent intensity of the fluorescein to diminish due to quenching.
Lysozyme is a naturally occurring protein that can be found in chicken egg as well as in human tears and saliva. The enzyme has antibacterial properties to gram-positive bacteria, which helps ward off bacteria that cause diseases like salmonella. Our device is designed to detect a protein target, i.e. lysozyme, using a DNA aptamer. Upon detection, the complementary strand to the DNA aptamer is released and the flurophore is free to fluoresce. The main reason that we chose to use lysozyme protein over other options is because it is readily availabe for our team to experiment with. We aim to build a multi-aptamer nanoparticle system that can detect viral proteins in the future; however, we first aim to understand the mechanism of a single aptamer device that detects an easily accessible protein. The current application of our device would be to detect the amount of lysozyme in a certain food sample.
Fluorescent DNA Lysozyme Detector: Mechanism
1. Stoltenburg, Regina, Christine Reinemann, and Beate Strehlitz. "SELEX? A (r) evolutionary method to generate high-affinity nucleic acid ligands." Biomolecular engineering 24.4 (2007): 381-403.
2. Jayasena, Sumedha D. "Aptamers: an emerging class of molecules that rival antibodies in diagnostics." Clinical Chemistry 45.9 (1999): 1628-1650.