Team:UC Davis/Electrochemistry

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   <h2>Protein Engineering</h2>
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   <h2>Electrode Choice</h2>
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Having settled on NAD<sup>+</sup> 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.
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<p>Electrode Choice</p>
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We developed our Electrode System to be:
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Sensitive: have  a low limit of detection for NADH
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Reactive: Detect NADH with high linear range
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Selective: Be robust to any possible solution components
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Affordable: Cost accessible to the average consumer
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Efficient: Use a low sample volume
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Compatible: Be compatible with our, as well as other potentiostats
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Portable
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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, <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Electrode_Choice" class="brightlink">click here</a>.
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_System_Optimization">
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<p>System Optimization</p>
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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.
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<p>To find more about our system optimization,
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_System_Optimization" class="brightlink">
 +
click here
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</a>.
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</p>
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
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<div class="mainTitleHeader">
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<p>Enzyme Dependent Activity</p>
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<p align="center"><img src="https://static.igem.org/mediawiki/2014/b/bb/EnzymeTESTTTT.png" width="650px" style="border:3px solid black" class="genpic"></p>
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<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.
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</p>
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<p>
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To find out more about our enzyme testing,
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests" class="brightlink">
 +
click here
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</a>.
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Latest revision as of 02:51, 18 October 2014

UC Davis iGEM 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 .

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