This project for the IGEM competition is geared towards creating a system that detects chemicals by way of scent for potential health and military use. Current state-of-the-art inorganic hardware sensors for biological and chemical agent detection are highly tailored for specific chemicals and find difficulty when used to detect compounds outside of a highly defined analyte set. Olfactory receptors are G Protein Coupled Receptors (GPCRs) that discriminate thousands of odorants based on genetic sequences that in the presence of a ligand cause cells to generate an electric potential that is measurable using microelectrode arrays (MEAs). Here, we modify HT-22 cells by adding individual olfactory receptors plasmids via nucleofection. Two systems were developed to measure the action potential of neurons: 1) a bioreactor was designed with a peristaltic pump system allowing for media to flow across a MEA cultured with neurons which enables the controlled addition of liquid samples for action potential measurement, and 2) a photosensor controlled by an Arduino system that measures fluorescence over a cell chamber with constant airflow. Analysis of modified neurons serve as a representative model for exploiting the sensitivity and selectivity of native olfactory systems to be used as rapid detection systems for applications in security, medical, and health capacities.
USMA West Point's "eNOSE" was designed with the purpose of detecting improvised explosive devices (IED's), a threat to the present-day Warfighter in Iraq and Afghanistan. Alternatives are bomb sniffing dogs and ground penetrating radar, but many small inconsistencies cause these options to be somewhat unreliable. "eNOSE" is a biosensor that uses the same biological machinery as a dog, but without the same inconsistencies. The device uses olfaction, or the sense of smell, to detect odorants in a highly specific and sensitive manner. "eNOSE" is here to change the battlefield, security, and medicine - it is here to "detect the undetectable."
DESCRIPTION
Cell receptors that use secondary messenger pathways encoded from genetic sequences offer sensitive and specific detection of agents (based on detection of organic functional groups, lipids, sugars, nucleotides, and proteins) that are produced through organic synthesis or through biological means such as viruses, bacteria, spores, and biological toxins. Olfactory receptors generate action potentials that can be measured with current photonic and electrical systems. This form of chemoreception can improve the narrow range of current inorganic hardware sensors and limit high false positive/negative rates because using a broad range of encoded genes (even from different species) will specifically detect almost any biological agent.
We propose to create a sensor platform technology based on the most specific sensation method: chemoreceptors. We will use currently existing technology to measure the rapid and effective transduction signal associated with the binding of a ligand to a cell receptor that leads to a measureable action potential. The binding of a ligand, such as an odorant, to a cell surface receptor initiates a cascade of enzymatic reactions to produce a secondary messenger to open an ion channel producing an influx of cations (normally Ca2+ and Na+) depolarizing the cell. This change in electric potential is a measureable transduction using current microelectrode arrays (MEAs) and fluorescent voltage-sensitive (FVS) dyes such as calcium indicators.
Previous identification hardware sensors are too slow and bulky for rapid response detect-to-protect applications. Rapid diagnostic tests based on genetically engineered B cells have been produced with a CANARY bioelectric sensor that emit a photon in response to a specific bio-agent bound to membrane-bound antibodies. Even though CANARY provides aerosol collection, the detector still requires a portable case, battery pack, and high speed moving centrifuge. Furthermore, current optical microresonators sensors based on small changes to refractive index are not specific enough yielding poor results. Not only do neurons contain the necessary hardware of G protein-coupled receptors to recognize individual ligands, they also offer the necessary physical transduction mechanism that is easily measurable with current cell physiological tools. The proposed system would require a few thousand cells that has a footprint on the order of square millimeters and require the proper nutrients to maintain survival of olfactory cells therefore eliminating consumables and complicated moving systems.
Introducing genetic sequences of olfactory receptors from across the animal kingdom will provide the opportunity to detect from a larger library of aerosolized materials. Furthermore, combing recombinatorial approaches will allow the ability to create genetic sequences to include the expression of unorthodox olfactory receptors to detect compounds of interest that are currently ‘odorless’. Recently, mammalian odorant receptors were expressed with recombinant adenovirus of a particular receptor gene to increase the number of sensory neurons. Leveraging this technology, we will create a platform in synthetic biology and genetic engineering to produce new cell lines or add individual receptors to current cells to improve their chemical specificity.
