Team:Aalto-Helsinki/Proof Of Concept
Metabolite Choice
As GutLux can only encounter luminal compounds, we had to consider this aspect when finalizing our target metabolite choices. Our chosen metabolites -- kynurenic acid and tryptamine -- were chosen not only because of their potential connections to mental health, but also because unlike other mental health-related metabolites, they are actually present within the gut lumen. For further justification on our chosen metabolites, please refer to the Choice of Metabolite section on the Design page.
Sensitivity and Specificity Analyses
After deciding on our Aryl hydrocarbon Receptor (AhR) system, we were particularly excited to use this in our biosensor as this system has been characterized in humans before. We were, however, faced with a few challenges with this system, most notably its ability to detect a variety of metabolites outside of our chosen two. This would be particularly helpful in the expansion of GutLux to detect other compounds, but we understood the need to optimize GutLux for our metabolites first. To address this, we looked at optimizing GutLux’s specificity.
When aiming to alter AhR to preferentially bind to kynurenic acid or tryptamine, our main difficulty came from the fact that the AhR protein structure has not been characterized yet. Hence, we decided to perform predictive analyses only at the binding domain of AhR -- the PAS-B domain. Experiments included a docking analysis and subsequent mutagenesis. To learn more about what we performed and our results, please refer to the Model page.
To observe that our biosensor was sensitive enough to detect kynurenic acid and tryptamine, we observed the biosensor's sensitivity and range using the Hill equation and given reported concentrations of the metabolites in previous literature. Further information on how this analysis was performed and what we observed can be found on the Model page.
Fluorescence Detection
To examine the cooperation of the biological and electrical system, we conducted practical research and developed a prototype capable of measuring the intensity of light with similar wavelength to our expected fluorescent signal, and sending this data through radio frequency (RF) transmittance. For the prototype, we researched the emission and excitation peaks of our chosen green fluorescent protein (GFP) and created a system that was capable of both mimicking and detecting the light at the specific wavelength. We also tested out the transmittance of the detected values from the light detecting system to a receiver system, from which we were able to collect a set of data of the light intensity through wireless communication. In addition to conducting practical research, we defined the properties and actual components for the final design. To find out more about the prototype and capsule design, please refer to our Hardware page.
Protein Detection
If we were able to continue on with our project, we would work to verify the proteins we produced. We would express both our AhR-ARNT-AIP (ARNT, Aryl hydrocarbon Receptor Nuclear Translocator; AIP, Aryl hydrocarbon Receptor Interacting Protein) and the GFP-containing plasmids and perform a Western Blot to ensure that the proteins observed were of the expected size.
Metabolite Concentrations
GutLux aims to provide researchers with the relative quantities of kynurenic acid and tryptamine, which is done by observing the intensity of fluorescence. As of the Wiki Freeze, we were unable to perform the following experiments, but this would be next in our proposed workflow.
After verifying our proteins, we would subject our biosensor cells to varying concentrations of kynurenic acid and tryptamine and observe the fluorescent light intensity. A standard curve would be mapped, with metabolite concentrations and intensity of fluorescence on separate axes. This would predict how much metabolite is present given a certain fluorescence observed.
GutLux In The Real World
Alongside optimizing GutLux components to fit the limitations and perform well in the real world, we met with experts to discuss what would be needed to put GutLux on the market for actual research use. After speaking with Rosa Tengvall from the Kasve company, we learned that GutLux would be classified as a type IIa medical device as defined by the EU Regulation of Medical Devices. Such a medical device has a list of safety considerations and requirements that must be completed. For further information on these requirements that GutLux would be required to fulfill, please refer to the Medical Device Regulation section on the Implementation page.
