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Team:UC Davis/Electrochemistry
From 2014.igem.org
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests"> | <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests"> | ||
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Sensitive: have a low limit of detection for NADH | Sensitive: have a low limit of detection for NADH | ||
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Reactive: Detect NADH with high linear range | Reactive: Detect NADH with high linear range | ||
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Selective: Be robust to any possible solution components | Selective: Be robust to any possible solution components | ||
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Affordable: Cost accessible to the average consumer | Affordable: Cost accessible to the average consumer | ||
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Efficient: Use a low sample volume | Efficient: Use a low sample volume | ||
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Compatible: Be compatible with our, as well as other potentiostats | Compatible: Be compatible with our, as well as other potentiostats | ||
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Portable | Portable | ||
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests"> | <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests"> | ||
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| - | <p> | + | <p>Enzyme Dependent Activity</p> |
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Having optimized our electrochemical setup, we were ready to measure enzyme activity. In order to establish system function, we tested a single enzyme and substrate over time. After corroborating system function with on enzyme, we tested our three engineered enzymes with twelve different substrates. Our electrochemical sensor was able to detect differences in enzyme activity at high levels of aldehyde, proving the viability of our system. Looking forward, we plan to further increase sensitivity of our electrochemical system in order to enhance our sensor’s capability. | Having optimized our electrochemical setup, we were ready to measure enzyme activity. In order to establish system function, we tested a single enzyme and substrate over time. After corroborating system function with on enzyme, we tested our three engineered enzymes with twelve different substrates. Our electrochemical sensor was able to detect differences in enzyme activity at high levels of aldehyde, proving the viability of our system. Looking forward, we plan to further increase sensitivity of our electrochemical system in order to enhance our sensor’s capability. | ||
Latest revision as of 02:51, 18 October 2014
Electrode Choice
Electrode Choice
System Optimization
System Optimization
Enzyme Dependent Actvity
Enzyme Dependent Actvity
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 .
Enzyme Dependent Activity
Having optimized our electrochemical setup, we were ready to measure enzyme activity. In order to establish system function, we tested a single enzyme and substrate over time. After corroborating system function with on enzyme, we tested our three engineered enzymes with twelve different substrates. Our electrochemical sensor was able to detect differences in enzyme activity at high levels of aldehyde, proving the viability of our system. Looking forward, we plan to further increase sensitivity of our electrochemical system in order to enhance our sensor’s capability.
To find out more about our enzyme testing, click here .
