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Team:UC Davis/Electrochemistry
From 2014.igem.org
Electrode Choice
Electrode Choice
System Optimization
System Optimization
Coupling Enzymes
Coupling Enzymes
Having settled on NAD+ dependent Aldehyde Dehydrogenases as our method of differentiating between aldehydes, we needed to develop an efficient electrode system to detect enzyme activity via NADH. We acquired, selected, and optimized an electrode setup for the detection of NADH at low concentrations in a complex solution. Additionally, we demonstrated the ability of the electrode setup to detect enzyme generated NADH over time, and thereby functionally deconvolute aldehyde profiles within a sample.
Electrode Choice
We developed our Electrode System to be:
- Sensitive: have a low limit of detection for NADH
- Reactive: Detect NADH with high linear range
- Selective: Be robust to any possible solution components
- Affordable: Cost accessible to the average consumer
- Efficient: Use a low sample volume
- Compatible: Be compatible with our, as well as other potentiostats
- Portable
We tested three screen printed base electrode types, and five different working electrode modification schemes in order to achieve the requisite sensitivity for our system. We settled on Dropsens screen printed #610 Electrodes, depicted above. To find out more about our electrode selection process, click here.
System Optimization
Once the electrode type was chosen on the basis of its sensitivity, the analytical solution components were optimized to maintain maximum selectivity for NADH. Any solution component introduced in the original Olive Oil Sample, aldehyde extraction processes, or the enzyme purification step could have theoretically impacted the electrode’s sensitivity for NADH. Collaboration with the protein engineering team led to the development of a solution mix that enabled selective detection of enzyme generated NADH in a solution with a large background of various solution components.
These interference is often shown in our graph as “NOISE”. In order to examine each and individual noise factor, we have divided our experiment into two parts:
Optimizing solution
Optimizing solution includes:
1) Testing how each and every chemical in the testing solution affects the signals collected from electrode.
2) Deciding on concentration of each chemical which would minimizing the noise level.
Optimizing system
This part of experiment deals with many internal and external possibilities that may or may not created the “noise” in our system. Some of our solutions has not shown any consistent improvement of decreasing these noise; however,few displays some improvement in getting rid of these noise.
To find more about our system optimization, click here.
Coupling Enzymes
Once enzymes have been engineered and successfully displayed their specificity, our mission is to come up with the most suitable solution and enzyme ratio to get the most clear signals from our electrode. This includes:
Measuring enzyme activity in our system:
Searching for satisfactory range of concentration for enzyme to be added to our solution in order to demonstrate the signals that are coherent to the enzyme kinetics data.
Demonstrate enzyme specificity measured from our system device:
Prove that our system and device display the same specificity of each enzyme as we previously seen on the enzyme kinetics data.
To find out more about our enzyme testing, click here.
