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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. Electrochemical systems are inherently sensitive to the addition of electroactive compounds to the solution being analyzed. In our biosensor, we needed to be able to combine an olive oil extraction solution containing aldehydes, purified protein, and buffer for our system to function properly. All of these system components had the potential to introduce electroactive solution components that would increase noise, or inhibit our system’s ability to see a signal related to NADH generation. Thus, it was necessary to optimize the protein purification as well as extraction buffers to allow unhindered electrode function. Additionally, we optimized the system to maintain a proper NADH signal to system noise ratio so as to allow for effective NADH detection and system function.
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.
